U.S. patent application number 13/699358 was filed with the patent office on 2013-06-13 for levocarrimycin, pharmaceutical compositions, preparation methods and uses thereof.
This patent application is currently assigned to Shenyang Toglian Group Co., Ltd.. The applicant listed for this patent is Yuyou Hao, Yang Jiang. Invention is credited to Yuyou Hao, Yang Jiang.
Application Number | 20130150316 13/699358 |
Document ID | / |
Family ID | 44975203 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130150316 |
Kind Code |
A1 |
Jiang; Yang ; et
al. |
June 13, 2013 |
LEVOCARRIMYCIN, PHARMACEUTICAL COMPOSITIONS, PREPARATION METHODS
AND USES THEREOF
Abstract
The present invention relates to levocarrimycin, its
pharmaceutical compositions, preparation methods and application.
Levocarrimycin is a mixture of isovalerylspiramycin III, II and I
as main components and contains some isobutyrylspiramycin III and
II, butyrylspiramycin III and II, propionylspiramycin III and II,
as well as acetylspiramycin III and II, among which, the content of
isovalerylspiramycin III is no less than 30 wt %, the total content
of isovalerylspiramycin III, II and I is no less than 60 wt %, and
the content of acylspiramycin is 80-98 wt %. Specific optical
rotation of said levocarrimycin is
[.alpha.].sub.D=-52.degree..about.-57.degree. in the solution of
0.02 g/ml chloroform at temperature of 25.degree. C. The present
invention also relates to the crystalline compound of
isovalerylspiramycin III, II or I in levocarrimycin, and
pharmaceutical compositions containing the said levocarrimycin. In
present invention, the active components in levocarrimycin or its
pharmaceutical compositions have optical activity and excellent
anti-infective effect.
Inventors: |
Jiang; Yang; (Liaoning,
CN) ; Hao; Yuyou; (Liaoning, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Jiang; Yang
Hao; Yuyou |
Liaoning
Liaoning |
|
CN
CN |
|
|
Assignee: |
Shenyang Toglian Group Co.,
Ltd.
Liaoning
CN
|
Family ID: |
44975203 |
Appl. No.: |
13/699358 |
Filed: |
May 25, 2011 |
PCT Filed: |
May 25, 2011 |
PCT NO: |
PCT/CN11/74658 |
371 Date: |
February 6, 2013 |
Current U.S.
Class: |
514/30 ;
435/76 |
Current CPC
Class: |
A61K 31/7048 20130101;
Y02A 50/483 20180101; A61K 31/7048 20130101; A61P 31/00 20180101;
C07H 17/08 20130101; Y02A 50/475 20180101; A61P 31/10 20180101;
Y02A 50/473 20180101; C12P 19/62 20130101; Y02A 50/478 20180101;
A61P 31/04 20180101; A61K 2300/00 20130101 |
Class at
Publication: |
514/30 ;
435/76 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; A61P 31/00 20060101 A61P031/00; A61P 31/04 20060101
A61P031/04; C12P 19/62 20060101 C12P019/62 |
Foreign Application Data
Date |
Code |
Application Number |
May 25, 2010 |
CN |
201010182027.9 |
Claims
1-21. (canceled)
22. A levocarrimycin, wherein said levocarrimycin is a mixture of
isovalerylspiramycin III, II and I as main components and contains
certain isobutyrylspiramycin III and II, butyrylspiramycin III and
II, propionylspiramycin III and II, as well as acetylspiramycin III
and II, among them the content of the isovalerylspiramycin III is
not less than 30 wt %, the total content of isovalerylspiramycin
III, II and I is no less than 60 wt %, and the content of
acylspiramycin is 80-98 wt %, preferably 85.about.98 wt %, further
preferably 90.about.98 wt % and most preferably 95.about.98 wt %;
the specific optical rotation of said levocarrimycin is
[.alpha.].sub.D=-52.degree..about.-57.degree. in the solution of
0.02 g/ml chloroform at temperature of 25.degree. C., preferably,
-54.degree..about.-56.degree. and further preferably,
-55.degree..
23. The levocarrimycin according to claim 22, wherein said
levocarrimycin also contains spiramycin III and other components,
among them the content of spiramycin III is no more than 1.0%, and
the total content of other components is 2.0.about.19 wt %,
preferably 2.0.about.14.0 wt %, further preferably 2.0.about.9.0 wt
% and most preferably 2.0.about.4.0 wt %.
24. The levocarrimycin according to claim 23, wherein said melting
point of said levocarrimycin is 112.about.122.degree. C.,
preferably 114.about.120.degree. C. and further preferably
116.about.118.degree. C.
25. The levocarrimycin according to claim 22, wherein said
isovalerylspiramycin III is a crystal compound of
levoisovalerylspiramycin III, the isovalerylspiramycin II is a
crystal compound of levoisovalerylspiramycin II, or the
isovalerylspiramycin I is a crystal compound of
levoisovalerylspiramycin I; when the isovalerylspiramycin III is a
crystal compound with III of levoisovalerylspiramycin, said crystal
compound measured by the X-ray powder diffraction with Cu--Ka ray
has characteristic peaks of 2.theta. at 8.0.degree., 10.0.degree.,
11.2.degree., 11.7.degree., 16.4.degree., 19.1.degree.,
19.6.degree., 20.0.degree., 21.4.degree., 22.9.degree.,
23.6.degree. and 29.4.degree.; when the isovalerylspiramycin II is
a crystal compound with II of levoisovalerylspiramycin, said
crystal compound measured by the X-ray powder diffraction with
Cu--Ka ray has characteristic peaks of 2.theta. at 10.0.degree.,
11.6.degree., 16.4.degree., 17.3.degree., 19.1.degree.,
21.2.degree., 22.1.degree., 22.7.degree., 26.4.degree.,
26.9.degree., 27.5.degree. and 31.5.degree.; when the
isovalerylspiramycin I is a crystal compound with I of
levoisovalerylspiramycin, said crystal compound measured by the
X-ray powder diffraction with Cu--Ka ray has characteristic peaks
of 2.theta. at 7.6.degree., 8.0.degree., 10.0.degree.,
11.4.degree., 16.4.degree., 17.0.degree., 17.5.degree.,
17.9.degree., 19.5.degree., 22.7.degree., 23.7.degree. and
24.4.degree..
26. A pharmaceutical composition of levocarrimycin, wherein said
pharmaceutical composition of levocarrimycin contains the
levocarrimycin according to claim 22 and pharmaceutically
acceptable carrier.
27. The pharmaceutical composition of levocarrimycin according to
claim 26, wherein the content of said levocarrimycin is 10.about.90
wt % of the pharmaceutical composition, preferably, 25.about.75 wt
%, and further preferably, 40.about.60 wt %.
28. The pharmaceutical composition of levocarrimycin according to
claim 27, wherein the pharmaceutical composition of levocarrimycin
exists in preparations applicable for pharmaceutical purpose, and
the preparations are liquid, solid, semisolid, or gas preparations,
said liquid preparations comprise injection, infusion solution,
solution, mixture, sirup, tincture, colloid, aromatic water,
glycerite, colloid solution, mucilage, suspension, or emulsion;
said solid preparations comprise power injection, lyophilized
powder injection, tablet, capsule, powder, granula, pill, sublimed
preparation, or pellicle; the semisolid preparations comprise
ointment, plaster, suppository, extract or gel; and the gas
preparations comprise aerosol or spray.
29. The pharmaceutical composition of levocarrimycin according to
claim 26, wherein the content of said levocarrimycin is
10.about.1500 mg per unit formulation, preferably, 100.about.1000
mg per unit formulation and further preferably, 200.about.500 mg
per unit dosage form.
30. A preparation method for levocarrimycin according to claim 22,
which includes culture, fermentation and extraction process,
wherein said culture and fermentation is: culture the cloned fungal
strains WSP-195 produced by spiramycin containing 4''-isovaleryl
transferase gene on an slant culture-medium, inoculate it in seed
medium, then inoculate it in fermentation medium after culturing,
and control the fermentation process by pH regulator; control the
pH at 6.0.about.9.0, preferably, 6.0.about.8.0, and further
preferably, 6.0.about.7.5; the variation curves for pH with time
show three continuous phases, the first phase satisfies formula
y.sub.1=k.sub.1x.sub.1+6.0, where
0.02275.ltoreq.k.sub.1.ltoreq.0.1364, 0<x.sub.1.ltoreq.22; the
second phase satisfies y.sub.2=k.sub.2x.sub.2+b.sub.2, where
-0.0735.ltoreq.k.sub.2.ltoreq.0, 6.5<b.sub.2.ltoreq.10.62,
22.ltoreq.x.sub.2.ltoreq.56; and the third phase satisfies formula
y.sub.3=k.sub.3x.sub.3+b.sub.3, where 0<k.sub.3.ltoreq.0.0078,
6.06.ltoreq.b.sub.3<6.5, 56.ltoreq.x.sub.3.ltoreq.120.
31. The preparation method according to claim 30, wherein said pH
regulator is at least one or combination of glucose, citric acid,
acetic acid, hydrochloric acid, ammonia, sodium hydroxide and
potassium hydroxide, preferably, one or combination of glucose,
citric acid, acetic acid and ammonia; further preferably, one or
combination of glucose and ammonia.
32. The preparation method according to claim 30, wherein said
extraction process is: process the fermentation liquor with
aluminum sulfate to obtain the filtrate, adjust pH to
8.5.about.9.0, extract with butyl acetate, wash the butyl acetate
extract with non-saline water and 1% NaH.sub.2PO.sub.4
respectively, then extract with water of pH 2.0.about.2.5 to obtain
aqueous extract, adjust the pH to 4.5.about.5.5, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.5.about.9.0, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
33. The preparation method according to claim 30, wherein said
slant culture-medium contains 2% soybean cake meal, 1% glucose, 3%
starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar.
34. The preparation method according to claim 30, wherein said seed
medium contains 1.5% soybean cake meal, 3.0% starch, 0.4% NaCl,
0.5% CaCO.sub.3, 0.3% peptone and 0.05% KH.sub.2PO.sub.4.
35. The preparation method according to claim 30, wherein said
fermentation medium contains 0.5% glucose, 6.0% starch, 0.5% yeast
powder, 2.0% fish meal, 0.6% NH.sub.4NO.sub.3, 1.0% NaCl, 0.5%
CaCO.sub.3, 0.05% KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean
oil and 0.02% defoaming agent.
36. The preparation method according to claim 30, wherein the
culture on the slant culture-medium lasts for 8.about.15 days at
temperature of 28.about.38.degree. C.; the culture on the seed
medium lasts for 40.about.80 hours at temperature of
25.about.30.degree. C.; and the fermentation on the fermentation
medium lasts for 72.about.120 hours at temperature of
26.about.30.degree. C.
37. The preparation method according to claim 30, wherein said
preparation method also comprises the following steps: a) Separate
and purify the levocarrimycin to obtain levoisovalerylspiramycin I,
II or III; b) Recrystallize the levoisovalerylspiramycin I, II or
III to obtain the crystal compound of levoisovalerylspiramycin I,
II or III; c) Eliminate the acetonitrile in the residual
levocarrimycin after separating and purifying
levoisovalerylspiramycin I, II or III in step a) through rotary
evaporation, then extract with 1 time amount of ethyl acetate, and
eliminate the ethyl acetate in the extract through rotary
evaporation to obtain paste sample; re-dissolve the obtained sample
with petroleum ether, and eliminate the petroleum ether through
rotary evaporation to obtain the levocarrimycin; d) Mix the crystal
compound of levoisovalerylspiramycin I, II or III obtained in step
b) with the levocarrimycin obtained in step c) to obtain the
levocarrimycin, in which, the isovalerylspiramycin I, II or III is
the crystal compound of levoisovalerylspiramycin I, II or III.
38. The preparation method according to claim 37, wherein said
separation and purification in step a) is: Purify the
levocarrimycin obtained in the preliminary separation with a
preparative high performance liquid chromatography, prepare
chromatographic column with ODS, use acetonitrile and ammonium
acetate buffer solution as the mobile phase in a gradient elute;
record the separated UV spectrogram through UV detection, and
collect the target peaks of levoisovalerylspiramycin I, II or III
components: Chromatographic column: ODS preparative chromatographic
column; Mobile phase: acetonitrile (A), 100 mM ammonium acetate
solution (B); Gradient condition: adopting linear gradient for
0.about.60 min, A is 25%-65%; and 61.about.90 min, A is
65%.about.90%; Flow velocity: 260 mL/min; Injection volume: 10 mL;
Sampling concentration: 0.5 g/mL; Measurement wavelength: 231 nm;
Way of collecting: collection via UV triggering; Collect the sample
of levoisovalerylspiramycin I according to the retention time
44.759 min of levoisovalerylspiramycin I; or collect the sample of
isovalerylspiramycin II according to the retention time 43.34 min
of isovalerylspiramycin II; or collect the sample of
levoisovalerylspiramycin III according to the retention time 48.009
of levoisovalerylspiramycin III; then eliminate acetonitrile
through rotary evaporation, extract with 1 time amount of ethyl
acetate, and eliminate the ethyl acetate in the extract through
rotary evaporation to obtain paste sample; re-dissolve the obtained
sample with petroleum ether, and eliminate the petroleum ether
through rotary evaporation to obtain the white solid powder of
levoisovalerylspiramycin I, II or III.
39. The preparation method according to claim 38, wherein when the
isovalerylspiramycin I is a crystal compound of
levoisovalerylspiramycin I, the crystal compound is obtained
through the following recrystallization process: dissolve the white
solid powder of levoisovalerylspiramycin I in the mixed solvent of
absolute methanol, absolute ethyl alcohol and anhydrous acetone,
then add pure water while stirring, after that, reduce the
temperature to 5.degree. C..about.15.degree. C. while stirring
continuously, to obtain the crystal compound of the
levoisovalerylspiramycin I, in which the volume ratio of ethyl
acetate to absolute ethyl alcohol to anhydrous acetone in the mixed
solvent is 1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1; when the isovalerylspiramycin II is a
crystal compound of levoisovalerylspiramycin II, the crystal
compound is obtained through the following recrystallization
process: dissolve the white solid powder of
levoisovalerylspiramycin II in the mixed solvent of absolute
methanol, absolute ethyl alcohol and anhydrous acetone, then add
pure water while stirring, after that, reduce the temperature to
5.degree. C..about.15.degree. C. while stirring continuously, to
obtain the crystal compound of the levoisovalerylspiramycin II, in
which the volume ratio of absolute methanol to absolute ethyl
alcohol to anhydrous acetone in the mixed solvent is
1:0.1.about.10:0.5.about.1, preferably, 1:2.about.8:0.8.about.1;
when the isovalerylspiramycin III is a crystal compound of
levoisovalerylspiramycin III, the crystal compound is obtained
through the following recrystallization process: dissolve the white
solid powder of levoisovalerylspiramycin III in the mixed solvent
of absolute methanol, absolute ethyl alcohol and anhydrous acetone,
then add pure water while stirring, after that, reduce the
temperature to 5.degree. C..about.15.degree. C. while stirring
continuously, to obtain the crystal compound of the
levoisovalerylspiramycin III, in which the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent is 1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of carrimycin raw
medicine and pharmaceutical preparations thereof, specifically to a
macrolide genetically-engineered antibiotic, in particular to
levocarrimycin, its preparation methods and uses in preparing drugs
for treating and preventing infectious diseases.
BACKGROUND OF THE INVENTION
[0002] Carrimycin is a new derivative of spiramycin developed by
adopting genetic engineering technology, which is originally named
biotechspiramycin and formerly named biotechmycin [Patent No.:
ZL97104440.6]. According to the "Rules for Chinese Approved Drug
Names", and upon technical review and confirmation of Chinese
Pharmacopoeia Commission, the Chinese generic name of
biotechspiramycin is changed to carrimycin.
[0003] Carrimycin is a fermentation product of
genetically-engineered bacteria. The chemical structure of
carrimycin mainly comprises 4''-isovalerylspiramycin, including
4''-isovalerylspiramycin I, II, III, and about 6 kinds of
4''-hydroxy acylated spiramycin, so the chemical name is
4''-acylspiramycin. Chemical structural form a of main component of
carrimycin is as shown in form a (1):
##STR00001## [0004] in which:
TABLE-US-00001 [0004] R R' Isovalerylspiramycin I H
COCH.sub.2CH(CH.sub.3).sub.2 Isovalerylspiramycin II COCH.sub.3
COCH.sub.2CH(CH.sub.3).sub.2 Isovalerylspiramycin III
COCH.sub.2CH.sub.3 COCH.sub.2CH(CH.sub.3).sub.2
[0005] Carrimycin is a 16-membered ring macrolide antibiotic, which
inhibits the protein synthesis by combining with ribosome of
bacterium.
[0006] In vitro test results show that carrimycin is effective
against gram-positive bacteria, especially some drug-resistance
bacteria such as .beta.-lactam resistance staphylococcus aureus and
erythrocin-resistance staphylococcus aureus, and has no significant
cross drug resistance with similar drugs. Meanwhile, carrimycin has
antibacterial activity for mycoplasma and chlamydia, as well as
some gram-negative bacteria, good antibacterial activity and tissue
permeability for epidemic toxoplasm and legionella, and still has
potential immunoregulation function. The antibacterial activity in
vivo is much better than that in vitro (ZL200310122420.9). Clinical
research shows that by taking carrimycin tablets 0.2-0.4 mg
everyday for 5.about.7 days, it is suitable for treating acute
bacterial pharyngitis and acute suppurative tonsillitis caused by
pyogenic streptococcus; bacterial nasosinusitis and acute
bronchitis caused by sensitized bacteria; mild pneumonia caused by
streptococcus pneumonia, haemophilus influenza and mycoplasma
pneumonia; nongonoccal urethritis caused by mycoplasma and
chlamydia; infectious diseases such as skin and soft tissue
infection, periodontitis and otitis media caused by sensitized
bacteria. The total effective rate is 92.68%. Carrimycin is safe
and effective.
[0007] Pharmacokinetics study shows that active components of
carrimycin are mainly isovalerylspiramycin I, II and III.
Carrimycin quickly metabolizes to spiramycin in vivo. According to
AUC.sub.0-t of parent drug isovalerylspiramycin I, II and II and
active metabolite spiramycin I, II and III, the absolute
bioavailability by oral administration is 91.6% averagely. It is
reported that the absolute bioavailability of spiramycin by oral
administration is 30.about.40% (Frydman A M et al J Antimicrob
Chemother. 1988, 22 (suppl B):93-103). It shows that the
isovalerylspiramycin significantly improves the bioavailability of
active component spiramycin. Single dose carrimycin is eliminated
slowly. T1/2 is between 23.about.27 hours.
[0008] Researches on the active components of carrimycin show that
a plurality of chiral carbon atoms exists in the molecular
structure of active components of carrimycin: isovalerylspiramycin
I, II and III. Chirality is a basic attribute of three-dimensional
body and one of the essential attributes of nature. Biological
macromolecules including protein, polysaccharide, nucleic acid and
enzyme as important basis of vital movement often have important
physiological functions. Chiral drug is a pair of enantiomers of
material object and mirror image obtained after molecular structure
of drug is introduced into the chiral center. These enantiomers are
basically the same regarding physicochemical properties but
different in optical rotation. The enantiomers are respectively
named R-type (dextrorotatory) or S-type (sinistral), and racemic.
In recent 20 years, as pharmaceutical research is more intensive,
it has been proved that the difference of drug enantiomer's
affinity with receptor caused by the difference of drug
enantiomer's stereoselectivity leads to great difference in
pharmacological action. Enantiomer with high activity among chiral
drugs is called eutomer; while the one with low or no activity is
called distomer. In many cases, the distomer not only has no
pharmacological action, but also offset that of eutomer. Sometimes,
severe toxic side reactions occur, showing the complexity of
difference in pharmacological function and determining great
difference in the therapeutic index of single enantiomer and the
racemate thereof. For example, the curative effect of well-known
DL-(+-) syntomycin is half of D (-) chloramphenicol; the
pharmaceutical activity of propranolol L-isomer is 100 times larger
than that of D-isomer; (-) adanon is a strong painkiller while (+)
is non-effective. There is also difference in toxicity. For
example, the two enantiomers of thalidomide have similar sedation
for mice, but only S (-) isomer and metabolin thereof have
embryotoxin and teratogenesis; ketamine is a widely used
anaesthetic and analgesic, but has side effects such as
hallucinating. Studies show that S(+) is 3.about.4 times more
effective than R(-) and toxic side effects have something to do
with the latter. The great difference of chiral drug's curative
effect has promoted the research and development of chiral drugs
and the development separation analysis. By using "chiral"
technology, we can remove those with no effect or toxic side
effects from drugs effectively and produce pure chiral drugs with
single and oriented structure, thus making more pure pharmaceutical
ingredients, further quickening curative effect and shortening the
course of treatment. Therefore, research on chiral drugs has become
one of the new methods for new medicine research worldwide.
National governments and pharmaceutical enterprises have invested
heavily in fields such as preparations of chiral drug, chiral
materials and chiral intermediate for research and development, for
the purpose of seizing dominance of chiral pharmacy market. Beside,
with continuous improvement of chiral technology, especially the
fast and wide use of liquid chromatography, the separation analysis
and determination of enantiomers of chiral drugs are promoted.
Chiral drugs of single enantiomer have been widely used.
[0009] Through a lot of researches on whether carrimycin has
optical activity too, the inventor is pleasantly surprised to find
that by adjusting and optimizing the culturing and fermentation
conditions, the inventor accidently gets a levocarrimycin with
optical rotation, which has better anti-infection activity.
Therefore, the present invention provides levocarrimycin,
preparation methods thereof and uses in preparing drugs for
preventing and treating infectious diseases.
SUMMARY OF THE INVENTION
[0010] The first object of the present invention is to provide
levocarrimycin, which has optical rotation and better
anti-infection activity at the same time.
[0011] The second object of the present invention is to provide a
pharmaceutical composition, the pharmaceutical composition contains
levocarrimycin with optical rotation provided by the present
invention and pharmaceutically acceptable carrier.
[0012] The third object of the present invention is to provide a
preparation method of levocarrimycin, the method is characterized
by simplified production process and easy-to-control quality
standard, and the prepared levocarrimycin has good effect, optical
rotation and better anti-infection activity.
[0013] The fourth object of the present invention is to provide a
uses of said levocarrimycin or said pharmaceutical composition of
levocarrimycin for manufacturing a medicament for the treatment and
prevention of infectious diseases. Especially, levocarrimycin or
pharmaceutical composition of levocarrimycin has good effect in
resistance to bacteria, chlamydia and mycoplasma and can be used as
drugs for infectious diseases.
[0014] To realize the first object of the prevent invention, the
following technical solutions are adopted:
[0015] A levocarrimycin, the levocarrimycin is a mixture of
isovalerylspiramycin III, II and I as main components and contains
certain content of isobutyrylspiramycin III and II,
butyrylspiramycin III and II, propionylspiramycin III and II, as
well as acetylspiramycin III and II, among them the content of the
isovalerylspiramycin III is no less than 30 wt %, the total content
of isovalerylspiramycin III, II and I is no less than 60 wt %, and
the content of acylspiramycin is 80-98 wt %, preferably 85.about.98
wt %, further preferably 90.about.98 wt % and most preferably
95.about.98 wt %; the specific optical rotation of said
levocarrimycin is [.alpha.]D=-52.degree..about.-57.degree. in the
solution of 0.02 g/ml chloroform at temperature of 25.degree. C.,
preferably, -54.degree..about.56.degree. and further preferably,
-55.degree..
[0016] The inventor has made a lot of researches on carrimycin. By
adjusting and optimizing the culture and fermentation conditions,
especially by strict controlling the pH during the fermentation
with pH regulator, the curves of pH variation with time show three
continuous phases and each phase satisfies certain formula
respectively, thus the levocarrimycin with optical activity is
obtained. The possible reason is that the content of components
with optical activity changed under the fermentation conditions or
the optical configuration changed under the fermentation
conditions.
[0017] Determination method of the specific optical rotation of
levocarrimycin in the present invention is: weigh levocarrimycin
prepared of present invention precisely, add chloroform solution
and dilute into solution of about 10 mg/ml; measure the optical
rotation by using D line of sodium spectrum (589.3 nm) with a
measuring length of 1 dm and a measuring temperature of 25.degree.
C. and using a verified polarimeter with accuracy of
0.0001.degree..
[0018] The melting range of levocarrimycin of the present invention
is 112.about.122.degree. C., preferably, 114.about.120.degree. C.,
further preferably, 116.about.118.degree. C.
[0019] The determination method of melting range is: put
appropriate amount of dried product in a capillary tube for melting
point determination; repeat the determination for 3 times and take
the average.
[0020] The levocarrimycin of the present invention has optical
rotation. According to modern pharmacological research, the
difference of drug enantiomer's affinity with receptor caused by
the difference of drug enantiomer's stereoselectivity leads to
great difference in pharmacological action. In vivo and in vitro
pharmacodynamic tests prove that the levocarrimycin of the present
invention has good anti-infection effect and good pharmacological
activity at the same time, thus providing a new drug for treating
infectious diseases and laying a foundation for researching chiral
pharmaceutical preparation of carrimycin.
[0021] In vivo and in vitro tests prove that the levocarrimycin of
the present invention has high sensitivity and low drug resistance,
Levocarrimycin not only is effective against drug-resistant
staphylococcus aureus, but also has inestimable value for bacterial
infection caused by antibiotic abuse. For example,
methicillin-resistant staphylococcus aureus (MRSA), Escherichia
coli infection caused by extended-spectrum .beta.lactamase (ESBL),
and infectious diseases caused by clostridium difficile (C-diff)
are all due to antibiotic abuse and are expected to be controlled
for the coming out of levocarrimycin.
[0022] The levocarrimycin also contains spiramycin III and other
components, among them the content of spiramycin III is no more
than 1.0%, and the total content of other components is
2.0.about.19 wt %, preferably 2.0.about.14.0 wt %, further
preferably 2.0.about.9.0 wt % and most preferably 2.0.about.4.0 wt
%. In the present invention, said other compositions contain at
least 3 improved homologs of spiramycin.
[0023] Levocarrimycin of the present invention is a mixture of
isovalerylspiramycin III, II and I as main components, among them
said isovalerylspiramycin III is a crystal compound of
levoisovalerylspiramycin III, said isovalerylspiramycin II is a
crystal compound of levoisovalerylspiramycin II, or said
isovalerylspiramycin I is a crystal compound of
levoisovalerylspiramycin I;
when the isovalerylspiramycin III is a crystal compound with III of
levoisovalerylspiramycin, said crystal compound measured by the
X-ray powder diffraction with Cu--K.alpha. ray has characteristic
peaks of 2.theta. at 8.0.degree., 10.0.degree., 11.2.degree.,
11.7.degree., 16.4.degree., 19.1.degree., 19.6.degree.,
20.0.degree., 21.4.degree., 22.9.degree., 23.6.degree. and
29.4.degree.; when the isovalerylspiramycin II is a crystal
compound with II of levoisovalerylspiramycin, said crystal compound
measured by the X-ray powder diffraction with Cu--K.alpha. ray has
characteristic peaks of 2.theta. at 10.0.degree., 11.6.degree.,
16.4.degree., 17.3.degree., 19.1.degree., 21.2.degree.,
22.1.degree., 22.7.degree., 26.4.degree., 26.9.degree.,
27.5.degree. and 31.5.degree.; when the isovalerylspiramycin I is a
crystal compound with I of levoisovalerylspiramycin, said crystal
compound measured by the X-ray powder diffraction with Cu--K.alpha.
ray has characteristic peaks of 2.theta. at 7.6.degree.,
8.0.degree., 10.0.degree., 11.4.degree., 16.4.degree.,
17.0.degree., 17.5.degree., 17.9.degree., 19.5.degree.,
22.7.degree., 23.7.degree. and 24.4.degree..
[0024] Through further research, the inventor finds that after
purifying and separating the levocarrimycin, single compositions of
isovalerylspiramycin III, II or I are obtained; recrystallize one
of the compositions, and the crystal compound of
isovalerylspiramycin III, II or I is obtained; mix one of the
crystal compounds with levocarrimycin to get levocarrimycin, in
which, the isovalerylspiramycin II, II or I is the crystal compound
of levoisovalerylspiramycin III, II, or I. In vivo pharmacodynamic
test proves that the pharmacological effect of levocarrimycin, in
which, the isovalerylspiramycin III, II or I is crystal of
levoisovalerylspiramycin III, II, or I is much better than that of
pure levocarrimycin.
[0025] To realize the second object of the prevent invention, the
following technical solutions are adopted:
[0026] A pharmaceutical composition of levocarrimycin in which the
pharmaceutical composition of levocarrimycin contains said
levocarrimycin and pharmaceutically acceptable carrier.
[0027] Among said pharmaceutical compositions of present invention,
the content of the levocarrimycin is of safe and therapeutically
effective amount and is 10.about.90 wt % of that of the
pharmaceutical composition, preferably, 25.about.75 wt %, and
further preferably, 40.about.60 wt %.
[0028] "Safe and therapeutically effective amount" as employed in
the present invention means the enough amount of drugs, compounds,
compositions, products or medicaments that could alleviate, reverse
or treat diseases of human and other mammals and that have no
severe harm to the tissues of mammals.
[0029] The terminology "Pharmaceutically acceptable carrier" of
present invention refers to conventional drug carriers in the field
of pharmacy, for example, diluents, excipients such as water,
fillers such as starch and cane sugar; adhesives such as cellulose
derivatives, alginate, gelatins and polyvinylpyrrolidone;
moistening agent such as glycerol; disintegrants such as agar,
calcium carbonate and sodium bicarbonate; absorption enhancer such
as quaternary ammonium compound; surfactants such as hexadecanol;
adsorptive carriers such as kaolin and bentonite; lubricants such
as talcum powder, calcium stearate, Mg and polyethylene glycol.
Besides, other adjuvants such as spices and sweeteners can also be
added in the pharmaceutical composition.
[0030] Compositions of the prevent invention can be any of
diluents, disintegrant, lubricants, filler, adhesive, humectants,
absorption enhancer, surfactant, excipient of safe and
therapeutically effective amount, or commonly used drug carrier of
safe and therapeutically effective amount in this field.
[0031] The pharmaceutical composition of levocarrimycin in the
present invention exists in preparations applicable for
pharmaceutical purpose, and the preparations are liquid, solid,
semisolid, or gas preparations.
[0032] The liquid preparations comprise injection, infusion
solution, solution, mixture, sirup, tincture or colloid,
[0033] The solid preparations comprise power injection, lyophilized
powder injection, tablet, capsule, powder, granula, pill, sublimed
preparation or pellicle;
[0034] The semisolid preparations comprise ointment, plaster,
suppository, extract or gel; The gas ones comprise aerosol or
spray.
[0035] The pharmaceutical composition of levocarrimycin in the
prevent invention, among them the content of said levocarrimycin is
10.about.1500 mg per unit formulation, preferably, 100.about.1000
mg per unit formulation and further preferably, 200.about.500 mg
per unit formulation.
[0036] To realize the third object of the prevent invention, the
following solutions are adopted:
[0037] A preparation method for levocarrimycin, which includes
culture, fermentation and extraction process, among them the
culture and fermentation is: culture the cloned fungal strains
WSP-195 produced by spiramycin containing 4''-isovaleryl
transferase gene on an slant culture-medium, inoculate it in a seed
medium, then inoculate it into fermentation medium after culturing,
and control the fermentation process by pH regulator. Fermentation
proceeds under the pH of 6.0.about.9.0, preferably, 6.0.about.8.0,
and further preferably, 6.0.about.7.5. The variation curves for pH
with time show three continuous phases, the first phase satisfies
formula y.sub.1=k.sub.1x.sub.1+6.0, where
0.0227.ltoreq.k.sub.1.ltoreq.0.1364, 0<x.sub.1.ltoreq.22; the
second phase satisfies y.sub.2=k.sub.2x.sub.2+b.sub.2, where
-0.0735.ltoreq.k.sub.2.ltoreq.0, 6.5<b.sub.2.ltoreq.10.62,
22.ltoreq.x.sub.2.ltoreq.56; and the third phase satisfies formula
y.sub.3=k.sub.3x.sub.3+b.sub.3, where
0.ltoreq.k.sub.3.ltoreq.0.0078, 6.06.ltoreq.b.sub.3.ltoreq.6.5,
56.ltoreq.x.sub.3.ltoreq.120.
[0038] In the present invention, by adjusting and optimizing the
culture and fermentation conditions, especially by controlling the
pH during the fermentation with pH regulator, the curves of pH
variation with time show three continuous phases and each phase
satisfies certain formula respectively, thus levocarrimycin with
optical activity is obtained.
[0039] In the present invention, the fermentation process is key,
the pH needs to be detected regularly during the whole fermentation
process and is controlled by adding pH regulator, in which, the pH
regulator is any one of or combination of glucose, citric acid,
acetic acid, hydrochloric acid, ammonia water, sodium hydroxide or
potassium hydroxide, preferably, glucose, citric acid, acetic acid,
ammonia water or combination thereof; further preferably, glucose,
ammonia water or combination thereof.
[0040] The preparation method of the present invention, in which
the extraction process is: process the fermentation liquor with
aluminum sulfate to obtain the filtrate, adjust pH to
8.5.about.9.0, extract with butyl acetate, wash the butyl acetate
extract with non-saline water and 1% NaH.sub.2PO.sub.4
respectively, then extract with water of pH 2.0.about.2.5 to obtain
aqueous extract, adjust the pH to 4.5.about.5.5, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.5.about.9.0, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
[0041] In said preparation method of the present invention, said
slant culture-medium contains 2% soybean cake meal, 1% glucose, 3%
starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar.
[0042] In said preparation method of the present invention, said
seed medium contains 1.5% soybean cake meal, 3.0% starch, 0.4%
NaCl, 0.5% CaCO.sub.3, 0.3% peptone and 0.05% KH.sub.2PO.sub.4.
[0043] In said preparation method of the present invention, said
fermentation medium contains 0.5% glucose, 6.0% starch, 0.5% yeast
powder, 2.0% fish meal, 0.6% NH.sub.4NO.sub.3, 1.0% NaCl, 0.5%
CaCO.sub.3, 0.05% KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean
oil and 0.02% defoaming agent.
[0044] In said preparation method of the present invention, the
culture on the slant culture-medium lasts for 8.about.15 days at
temperature of 28.about.38.degree. C.
[0045] In said preparation method of the present invention, the
culture on the seed medium lasts for 40-hours at temperature of
25.about.30.degree. C.
[0046] In said preparation method of the present invention, the
fermentation on the fermentation medium lasts for 72.about.120
hours at temperature of 26.about.30.degree. C.
[0047] When the levocarrimycin contains the crystal of
isovalerylspiramycin I, II or III, said preparation method also
comprises the following steps: [0048] a) Separate and purify the
levocarrimycin to obtain levoisovalerylspiramycin I, II or III;
[0049] b) Recrystallize the levoisovalerylspiramycin I, II or III
to obtain the crystal compound of levoisovalerylspiramycin I, II or
III; [0050] c) Eliminate the acetonitrile in the residual
levocarrimycin after separating and purifying
levoisovalerylspiramycin I, II or III in step a) through rotary
evaporation, then extract with 1 time amount of ethyl acetate, and
eliminate the ethyl acetate in the extract through rotary
evaporation to obtain paste sample; re-dissolve the obtained sample
with petroleum ether, and eliminate the petroleum ether through
rotary evaporation to obtain the levocarrimycin; [0051] d) Mix the
crystal compound of levoisovalerylspiramycin I, II or III obtained
in step b) with the levocarrimycin obtained in step c) to obtain
the levocarrimycin, among which, the isovalerylspiramycin I, II or
III is the crystal compound of levoisovalerylspiramycin I, II or
III.
[0052] The preparation method of present invention, said separation
and purification process in step a) is: Purify the levocarrimycin
obtained in the preliminary separation with a preparative high
performance liquid chromatography, prepare chromatographic column
with ODS, use acetonitrile and ammonium acetate buffer solution as
the mobile phase in a gradient elute; record the separated UV
spectrogram through UV detection, and collect the target peaks of
levoisovalerylspiramycin I, II or III components:
Chromatographic column: ODS preparative chromatographic column;
[0053] Mobile phase: acetonitrile (A), 100 mM ammonium acetate
solution (B);
Gradient condition: adopting linear gradient for 0.about.60 min, A
is 25%.about.65%; and 61.about.90 min, A is 65%-90%; Flow velocity:
260 mL/min; Injection volume: 10 mL; Sampling concentration: 0.5
g/mL; Measurement wavelength: 231 nm; Way of collecting: collection
via UV triggering;
[0054] Collect the sample of levoisovalerylspiramycin I according
to the retention time 44.759 min of levoisovalerylspiramycin I; or
collect the sample of isovalerylspiramycin II according to the
retention time 43.34 min of isovalerylspiramycin II; or collect the
sample of levoisovalerylspiramycin III according to the retention
time 48.009 of levoisovalerylspiramycin III; then eliminate
acetonitrile through rotary evaporation, extract with 1 time amount
of ethyl acetate, and eliminate the ethyl acetate in the extract
through rotary evaporation to obtain paste sample; re-dissolve the
obtained sample with petroleum ether, and eliminate the petroleum
ether through rotary evaporation to obtain the white solid powder
of levoisovalerylspiramycin I, II or III.
[0055] The preparation method of the present invention,
when the isovalerylspiramycin I is a crystal of
levoisovalerylspiramycin I, the crystal is obtained through the
following recrystallization process: dissolve the white solid
powder of levoisovalerylspiramycin I in the mixed solvent of
absolute methanol, absolute ethyl alcohol and anhydrous acetone,
then add pure water while stirring, after that, reduce the
temperature to 5.degree. C. 15.degree. C. while stirring
continuously, to obtain the crystal of the levoisovalerylspiramycin
I, in which, the volume ratio of ethyl acetate to absolute ethyl
alcohol to anhydrous acetone in the mixed solvent is
1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1;
[0056] Among them, the first preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin I is:
the volume of pure water added is 2.about.9 times of the sum of
ethyl acetate, absolute ethyl alcohol and anhydrous acetone,
preferably, 2.5.about.7.5 times; the speed in adding pure water is
4.about.10 ml/min, preferably, 6.about.8 ml/min.
[0057] The second preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin I is:
the volume ratio of ethyl acetate to absolute ethyl alcohol to
anhydrous acetone in the mixed solvent is
1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1.
[0058] The third preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin I is:
the stirring speed when adding pure water is 30.about.60 rpm,
preferably, 45.about.60 rpm; after the pure water is added, the
stirring speed is 10.about.30 rpm, preferably, 10.about.20 rpm.
[0059] The fourth preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin I is:
after pure water is added, the cooling speed is
1.about.3.quadrature. per hour, preferably, 1.about.1.5.quadrature.
per hour.
[0060] When the isovalerylspiramycin II is a crystal of
levoisovalerylspiramycin II, the crystal is obtained through the
following recrystallization process: dissolve the white solid
powder of levoisovalerylspiramycin II in the mixed solvent of
absolute methanol, absolute ethyl alcohol and anhydrous acetone,
then add pure water while stirring, after that, reduce the
temperature to 5.quadrature..about.15.quadrature. while stirring
continuously, to obtain the crystal of the levoisovalerylspiramycin
II, in which the volume ratio of absolute methanol to anhydrous
acetone to absolute ethyl alcohol in the mixed solvent is
1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1;
[0061] Among them, the first preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin II is:
the volume of pure water added is 2.about.9 times of the sum of
absolute methanol, absolute ethyl alcohol and anhydrous acetone,
preferably, 2.5.about.7.5 times; the speed in adding pure water is
4.about.10 ml/min, preferably, 6.about.8 ml/min.
[0062] The second preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin II is:
the volume ratio of absolute methanol to anhydrous acetone to
absolute ethyl alcohol in the mixed solvent is
1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1.
[0063] The third preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin II is:
the stirring speed when adding pure water is 30.about.60 rpm,
preferably, 45.about.60 rpm; after the pure water is added, the
stirring speed is 10.about.30 rpm, preferably, 10.about.20 rpm.
[0064] The fourth preferred technical solution for the
recrystallization of the crystal of levoisovalerylspiramycin II is:
after pure water is added, the cooling speed is
1.about.3.quadrature. er hour, preferably, 1.about.1.5.quadrature.
per hour.
[0065] When the isovalerylspiramycin III is a crystal of
levoisovalerylspiramycin III, the crystal compound is obtained
through the following recrystallization process: dissolve the white
solid powder of levoisovalerylspiramycin III in the mixed solvent
of absolute methanol, absolute ethyl alcohol and anhydrous acetone,
then add pure water while stirring, after that, reduce the
temperature to 5.quadrature..about.15.quadrature. while stirring
continuously, to obtain the crystal compound of the
levoisovalerylspiramycin III, in which the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent is 1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1;
[0066] Among them, the first preferred technical solution for the
recrystallization of the crystal compound of
levoisovalerylspiramycin III is: the volume of pure water added is
2.about.9 times of the sum of absolute methanol, absolute ethyl
alcohol and anhydrous acetone, preferably, 2.5.about.7.5 times; the
speed in adding pure water is 4.about.10 ml/min, preferably,
6.about.8 ml/min.
[0067] The second preferred technical solution for the
recrystallization of the crystal compound of
levoisovalerylspiramycin III is: the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent is 1:0.1.about.10:0.5.about.1, preferably,
1:2.about.8:0.8.about.1.
[0068] The third preferred technical solution for the
recrystallization of the crystal compound of
levoisovalerylspiramycin III is: the stirring speed when adding
pure water is 30.about.60 rpm, preferably, 45.about.60 rpm; after
the pure water is added, the stirring speed is 10.about.30 rpm,
preferably, 10.about.20 rpm.
[0069] The fourth preferred technical solution for the
recrystallization of the crystal compound of
levoisovalerylspiramycin III is: after pure water is added, the
cooling speed is 1.about.3.quadrature. per hour, preferably,
1.about.1.5.quadrature. per hour.
[0070] The present invention also provides uses of said
levocarrimycin or said pharmaceutical compositions for
manufacturing a medicament for the treatment of infectious
diseases.
[0071] In the present invention, said infectious diseases are these
diseases caused by infection of gram-positive bacterium,
staphylococcus aureus, streptococcus pneumoniae, mycoplasma
pneumoniae, chlamydia pneumoniae, ureaplasma urealyticum, Chlamydia
trachomatis, pyogenic streptococcus, Micrococcus catarrhalis,
gonococcus, bacillus influenzae, legionella or anaerobe.
[0072] The present invention further provides uses of said
levocarrimycin and said pharmaceutical composition for
manufacturing a antibacterial medicament, the bacteria include
streptococcus pneumoniae, Group A streptococcus, pyogenic
streptococcus, enterococcus, staphylococcus aureus, S. epidermids,
Catarrhal coccus, gonococcus, bacillus influenzae, escherichia
coli, enterotoxigenic escherichia coli, enteropathogenic
escherichia coli, enteroinvasive Escherichia coli, Pseudomonas
aeruginosa, Klebsiella pneumoniae, bacillus proteus vulgaris,
typhoid bacillus, acinetobacter, citrobacter, Serratia marcescens,
S. Sonnei, Sh. flexneri, Tritirachium album; legionella like
legionella pneumophila, legionella gormanii, legionella bozemanii,
legionella dumoffii, legionella jordanis, and legionella micdadei;
anaerobe like bacteroides fragilis, bacteroides thetaiotaomicron,
bacteroides vulgatus, bacteroides distasonis, bacteroides
prevotella, Prevotella asaccharolyticus, Prevotella oralis,
Fusobacteriumnu cleatum, Fusobacterium russll, bifidobacteria,
lactobacillus, peptostreptococcus, propionibacterium acnes,
clostridium perfringens, and yeast-like fungus.
[0073] Person skilled in the art usually know, that the amount of
active components necessary for treatment changes with various
factors, including the nature of disease and patient's age and
condition, and is finally determined by the doctor. If the
pharmaceutical compositions of levocarrimycin in the present
invention is administered by unit dosage form, the content of the
levocarrimycin is 10.about.1500 mg per unit dosage form,
preferably, 100.about.1000 mg per unit dosage form and further
preferably, 200.intg.500 mg per unit dosage form. The dose needed
everyday can be administered by single dose or divided dose.
[0074] In vitro pharmacodynamic test proves that the active
components in levocarrimycin or its pharmaceutical compositions
provided by present invention have optical activity and excellent
anti-infective effect. The active components not only have good
antibacterial activity against gram-positive bacterium, especially
staphylococcus aureus that is resistant to erythrocin,
.beta.-lactamase, streptococcus pneumonia, and pyogenic
streptococcus, but also are effective against some negative
bacteria such as catarrhal coccus, gonococcus, bacillus influenzae,
some legionella and anaerobe, especially mycoplasma pneumonia and
chlamydia pneumonia.
[0075] Compared with the prior art, the present invention has the
following advantages:
1) The levocarrimycin of the present invention has optical
rotation, however, according to the modern pharmacological
research, the difference of drug enantiomer's affinity with
receptor caused by the difference of drug enantiomer's
stereoselectivity leads to great difference in pharmacological
action. In vivo and in vitro pharmacodynamic tests prove that
levocarrimycin of the present invention has excellent
anti-infective effect and good pharmacological activity at the same
time, thus providing a new drug for curing infectious diseases and
laying a foundation for researching and developing chiral drugs of
carrimycin; in vivo pharmacodynamic test shows that the
levocarrimycin, in which the isovalerylspiramycin I, II or III is
the crystal compound of levoisovalerylspiramycin I, II or III has
better protection function for the curative effect of mice infected
by 12 strains of bacteria; 2) The preparation method for
levocarrimycin provided in the present invention, by adjusting and
optimizing the culture and fermentation conditions, especially by
controlling the pH during the fermentation with pH regulator, the
curves of pH variation with time show three continuous phases and
each phase satisfies certain formula respectively, thus the
levocarrimycin with optical activity is obtained; 3) The
preparation method for levocarrimycin provided in the present
invention, which is featured by simplified production process, is
suitable for large-scale industrial production.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG. 1 is the variation curve of pH value-time during the
fermentation in example 1 of the present invention;
[0077] FIG. 2 is the variation curve of pH value-time during the
fermentation in example 2 of the present invention;
[0078] FIG. 3 is the variation curve of pH value-time during the
fermentation in example 3 of the present invention;
[0079] FIG. 4 is the liquid chromatogram of components of standard
carrimycin, among them, [0080] 1--spiramycin III [0081]
2--monoacetyl spiramycin II [0082] 3--monoacetyl spiramycin III
[0083] 4--propionylspiramycin II [0084] 5--propionylspiramycin III
[0085] 6--(iso-) butyrylspiramycin II [0086]
7--isovalerylspiramycin I [0087] 8--(iso-) butyrylspiramycin III
[0088] 9--isovalerylspiramycin II [0089] 10--isovalerylspiramycin
III
[0090] FIG. 5 is the liquid chromatogram of levocarrimycin provided
in example 4 of the present invention.
[0091] FIG. 6 is the X-ray powder diffraction pattern of crystal
compound of levoisovalerylspiramycin I in the present
invention;
[0092] FIG. 7 is the X-ray powder diffraction pattern of crystal
compound of levoisovalerylspiramycin II in the present
invention;
[0093] FIG. 8 is the X-ray powder diffraction pattern of crystal
compound of levoisovalerylspiramycin III in the present
invention.
DETAILED DESCRIPTION OF PREFERRED EXAMPLES
[0094] The followings are the examples of the present invention and
these examples aim to further describe, rather than limit, the
present invention.
Example 1
Preparation of Levocarrimycin
1) Culture and Fermentation
[0095] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on an slant
culture-medium, inoculate it in seed medium, then inoculate it in
fermentation medium after culturing, and control the fermentation
process by glucose and ammonia water. The fermentation lasts for
120 h under the pH value is 6.0.about.9.0. The variation curves for
pH with time show three continuous phases, the first phase
satisfies formula y.sub.1=0.1364x.sub.1+6.0, where
0<x.sub.1.ltoreq.22; the second phase satisfies formula
y.sub.2=-0.0735x.sub.2+10.64, where 22.ltoreq.xhd 2.ltoreq.56; the
third phase satisfies formula y.sub.3=0.0078x.sub.3+6.06, where
56.ltoreq.x.sub.3.ltoreq.120, see FIG. 1 for the variation curve,
obtain the fermentation broth.
2) Extraction
[0096] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 9.0, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.5 water to
obtain aqueous extract, adjust the pH to 4.5, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.5, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 2
Preparation of Levocarrimycin
1) Culture and Fermentation
[0097] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on an slant
culture-medium, inoculate it in seed medium, then inoculate it in
fermentation medium after culture, and control the fermentation
process by glucose and sodium hydroxide. The fermentation lasts for
110 h under the condition of the pH value is 6.0.about.8.0. The
variation curves for pH with time show three continuous phases, the
first phase satisfies formula y.sub.1=0.0909x.sub.1+6.4, where
0<x.sub.1.ltoreq.22; the second phase satisfies formula
y.sub.2=-0.0441x.sub.2+7.8, where 22.ltoreq.x.sub.2.ltoreq.56; and
the third phase satisfies formula y.sub.3=0.0078x.sub.3+6.06, where
56.ltoreq.x.sub.3.ltoreq.110, see FIG. 2 for the variation curve,
obtain the fermentation broth.
2) Extraction
[0098] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 8.9, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.2 water to
obtain aqueous extract, adjust the pH to 4.2, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.5, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 3
Preparation of Levocarrimycin
1) Culture and Fermentation
[0099] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on an slant
culture-medium, inoculate it in seed medium, then inoculate in
fermentation medium after culture, and control the fermentation
process by glucose and citric acid. The fermentation lasts for 115
h under the condition of the pH value is 6.0.about.7.5. The
variation curves for pH with time show three continuous phases, the
first phase satisfies formula y.sub.1=0.0682x.sub.1+6.0, where
0<x.sub.1<22; the second phase satisfies formula
y.sub.2=-0.0294x.sub.2+8.147, where 22<x.sub.2<56; the third
phase satisfies formula y.sub.3=0.0078x.sub.3+6.06, where
56<x.sub.3<115, see FIG. 3 for the variation curve, obtain
the fermentation broth.
2) Extraction
[0100] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 8.6, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.3 water to
obtain aqueous extract, adjust the pH to 5.2, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.7, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 4
Preparation of Levocarrimycin
1) Culture and Fermentation
[0101] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on a slant
culture-medium which containing 2% soybean cake meal, 1% glucose,
3% starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar for 15 days at
28.degree. C., inoculate it in seed medium which containing 1.5%
soybean cake meal, 3.0% starch, 0.4% NaCl, 0.5% CaCO.sub.3, 0.3%
peptone and 0.05% KH.sub.2PO.sub.4 for 80 hours at 25.degree. C.,
then inoculate in fermentation medium which containing 0.5%
glucose, 6.0% starch, 0.5% yeast powder, 2.0% fish meal, 0.6%
NH.sub.4NO.sub.3, 1.0% NaCl, 0.5% CaCO.sub.3, 0.05%
KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean oil and 0.02%
defoamer with a inoculation size of 0.1%, and control the
fermentation process by glucose and ammonia water. The fermentation
lasts for 120 h under the pH value is 6.0.about.9.0. The variation
curves for pH with time show three continuous phases, the first
phase satisfies formula y.sub.1=0.1364x.sub.1+6.0, where
0<x1.ltoreq.22; the second phase satisfies formula
y.sub.2=-0.0735x.sub.2+10.64, where 22.ltoreq.x.sub.2.ltoreq.56;
the third phase satisfies formula y.sub.3=0.0078x.sub.3+6.06, where
56.ltoreq.x.sub.3.ltoreq.120, and the fermentation broth is
obtained.
2) Extraction
[0102] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 8.5, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.0 water to
obtain aqueous extract, adjust the pH to 4.5, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.5 to obtain precipitate, wash
precipitate with purified water dry it to obtain
levocarrimycin.
Example 5
Preparation of Levocarrimycin
1) Culture and Fermentation
[0103] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on a slant
culture-medium which containing 2% soybean cake meal, 1% glucose,
3% starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar for 8 days at
38.degree. C., inoculate it in seed medium which containing 1.5%
soybean cake meal, 3.0% starch, 0.4% NaCl, 0.5% CaCO.sub.3, 0.3%
peptone and 0.05% KH.sub.2PO.sub.4 for 40 hours at 30.degree. C.,
then inoculate in fermentation medium which containing 0.5%
glucose, 6.0% starch, 0.5% yeast powder, 2.0% fish meal, 0.6%
NH.sub.4NO.sub.3, 1.0% NaCl, 0.5% CaCO.sub.3, 0.05%
KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean oil and 0.02%
defoamer with a inoculation size of 20%, and control the
fermentation process by glucose and ammonia water. The fermentation
lasts for 115 h under the pH value is 6.0.about.7.5 at
30.quadrature.. The variation curves for pH with time show three
continuous phases, the first phase satisfies formula
y.sub.1=0.0682x.sub.1+6.0, where 0<x.sub.1.ltoreq.22; the second
phase satisfies formula y.sub.2=-0.0294x.sub.2+8.147, where
22.ltoreq.x.sub.2.ltoreq.56; the third phase satisfies formula
y.sub.3=0.0078x.sub.3+6.06, where 56.ltoreq.x.sub.3.ltoreq.115, see
FIG. 3 for the variation curve, obtain the fermentation broth.
2) Extraction
[0104] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 9.0, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.5 water to
obtain aqueous extract, adjust the pH to 4.5-5.5, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 9.0, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 6
Preparation of Levocarrimycin
1) Culture and Fermentation
[0105] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on a slant
culture-medium which containing 2% soybean cake meal, 1% glucose,
3% starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar for 12 days at
30.degree. C., inoculate it in seed medium which containing 1.5%
soybean cake meal, 3.0% starch, 0.4% NaCl, 0.5% CaCO.sub.3, 0.3%
peptone and 0.05% KH.sub.2PO.sub.4 for 60 hours at 28.quadrature.,
then inoculate in fermentation medium which containing 0.5%
glucose, 6.0% starch, 0.5% yeast powder, 2.0% fish meal, 0.6%
NH.sub.4NO.sub.3, 1.0% NaCl, 0.5% CaCO.sub.3, 0.05%
KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean oil and 0.02%
defoamer with a inoculation size of 10%, and control the
fermentation process by glucose and ammonia water. The fermentation
lasts for 90 h under the pH value is 6.0.about.7.5 at
28.quadrature.. The variation curves for pH with time show three
continuous phases, the first phase satisfies formula
y.sub.1=0.0682x.sub.1+6.0, where 0<x.sub.1.ltoreq.22; the second
phase satisfies formula y.sub.2=-0.0294x.sub.2+8.147, where
22.ltoreq.x.sub.2.ltoreq.56; the third phase satisfies formula
y.sub.3=0.0078x.sub.3+6.06, where 56.ltoreq.x.sub.3.ltoreq.90, see
FIG. 3 for the variation curve, obtain the fermentation broth.
2) Extraction
[0106] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 8.7, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.2 water to
obtain aqueous extract, adjust the pH to 5.0, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.7, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 7
Preparation of Levocarrimycin
1) Culture and Fermentation
[0107] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on a slant
culture-medium which containing 2% soybean cake meal, 1% glucose,
3% starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar for 10 days at
35.quadrature., inoculate it in seed medium which containing 1.5%
soybean cake meal, 3.0% starch, 0.4% NaCl, 0.5% CaCO.sub.3, 0.3%
peptone and 0.05% KH.sub.2PO.sub.4 for 55 hours at 26.quadrature.,
then inoculate in fermentation medium which containing 0.5%
glucose, 6.0% starch, 0.5% yeast powder, 2.0% fish meal, 0.6%
NH.sub.4NO.sub.3, 1.0% NaCl, 0.5% CaCO.sub.3, 0.05%
KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean oil and 0.02%
defoamer with a inoculation size of 15%, and control the
fermentation process by glucose and ammonia water. The fermentation
lasts for 115 h under the pH value is 6.0.about.7.5 at
27.quadrature.. The variation curves for pH with time show three
continuous phases, the first phase satisfies formula
y.sub.1=0.0682x.sub.1+6.0, where 0<x.ltoreq.22; the second phase
satisfies formula y.sub.2=-0.0294x.sub.2+8.147, where
22.ltoreq.x.sub.2.ltoreq.56; the third phase satisfies formula
y.sub.3=0.0078x.sub.3+6.06, where 56.ltoreq.x.sub.3.ltoreq.110, see
FIG. 3 for the variation curve, obtain the fermentation broth.
2) Extraction
[0108] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 8.6, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.3 water to
obtain aqueous extract, adjust the pH to 4.8, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.8, obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 8
Preparation of Levocarrimycin
1) Culture and Fermentation
[0109] Culture the cloned fungal strains WSJ-195 produced by
spiramycin containing 4''-isovaleryl transferase gene on a slant
culture-medium which containing 2% soybean cake meal, 1% glucose,
3% starch, 0.5% CaCO.sub.3, 0.4% NaCl and 2% agar for 13 days at
36.quadrature., inoculate it in seed medium which containing 1.5%
soybean cake meal, 3.0% starch, 0.4% NaCl, 0.5% CaCO.sub.3, 0.3%
peptone and 0.05% KH.sub.2PO.sub.4, for 75 hours at 27.quadrature.,
then inoculate in fermentation medium which containing 0.5%
glucose, 6.0% starch, 0.5% yeast powder, 2.0% fish meal, 0.6%
NH.sub.4NO.sub.3, 1.0% NaCl, 0.5% CaCO.sub.3, 0.05%
KH.sub.2PO.sub.4, 0.1% MgSO.sub.4, 0.5% soybean oil and 0.02%
defoamer with a inoculation size of 0.5%, and control the
fermentation process by glucose and ammonia water. The fermentation
lasts for 98 h under the pH value is 6.0.about.8.0 at
29.quadrature.. The variation curves for pH with time show three
continuous phases, the first phase satisfies formula
y.sub.1=0.0909x.sub.1+6.4, where 0<x.sub.1.ltoreq.22; the second
phase satisfies formula y.sub.2=-0.0441x.sub.2+7.8, where
22.ltoreq.x.sub.2.ltoreq.56; the third phase satisfies formula
y.sub.3=0.0078x.sub.3+6.06, where 56.ltoreq.x.sub.3.ltoreq.110, see
FIG. 2 for the variation curve, obtain the fermentation broth.
2) Extraction
[0110] Process the fermentation broth with aluminum sulfate to
obtain filtrate, adjust the pH to 8.9, extract with butyl acetate,
wash the butyl acetate extract with non-saline water and 1%
NaH.sub.2PO.sub.4 respectively, then extract with pH 2.4 water to
obtain aqueous extract, adjust the pH to 4.6, volatilize and
eliminate the residual butyl acetate to obtain hydrous extract,
filter and adjust the pH to 8.6 to obtain precipitate, wash
precipitate with purified water and dry it to obtain
levocarrimycin.
Example 9
HPLC Quantitative Determination Method of Levocarrimycin
[0111] Determine by high performance liquid chromatography
(Appendix V D of Chinese Pharmacopoeia 2005 (2))
[0112] Adopt Venusil XBP C18 (L) 150{acute over (.orgate.)} (200
mm.times.4.6 mm, 5 um) chromatographic column (AGELA TECHNOLOGIES),
mobile phase A is acetonitrile, mobile phase B is 0.01 molL.sup.-1
ammonium acetate solution (adjust the pH value to 7.0 by ammonia
water), gradient elution according to the following table; the
wavelength is 232 nm, the flow rate is 1.0 mLmin.sup.-1, the column
temperature is 25.quadrature. and the injection volume is 20
.mu.l.
TABLE-US-00002 Mobile phase A Mobile phase B Time (min) (%) (%) 0
35 65 15 50 50 50 65 35 51 35 65 70 35 65
[0113] The chromatographic condition and system suitability test
shall refer to the liquid chromatogram of standard carrimycin
component (FIG. 4). Adjusting the chromatographic condition and, if
necessary, change the gradient elution condition of mobile phase to
make the sample levocarrimycin component consistent with spectra of
standard carrimycin component (FIG. 4).
[0114] Solution of standard sample: weigh proper amount of standard
product accurately, dilute it by the mixed liquor of 0.01 mol/L
ammonium acetate solution (adjust the pH value to 7.0 by ammonia
water) and acetonitrile in a proportion of 65:35 to a concentration
of 0.4 mg/ml-0.6 mg/ml as the solution of standard sample and shake
well for future use.
[0115] Solution of testing sample: weigh 50 mg testing sample,
dilute it by the mixed liquor of 0.01 mol/L ammonium acetate
solution (adjust the pH value to 7.0 by ammonia water) and
acetonitrile (proportion of 65 to 35) to 50 ml as the solution of
sample product and shake well for future use. Calculate based on
the peak area of isovalerylspiramycin III by external standard
method. The isovalerylspiramycin III shall not be less than 30%,
the isovalerylspiramycin (I+II+III) shall not be less than 60%; the
total content of acylation spiramycin's 9 components shall not be
less than 80%, the amount of spiramycin III shall not be more than
1.0% and the total content of other unknown components shall not be
more than 5.0%. The calculation formula is shown as follows:
Isovalerylspiramycin III (%)=A.sub.isovaleryl
III.times.W.sub.S.times.P/(A.sub.S.times.W.sub.T).times.100%
Isovalerylspiramycin (I+II+III) (%)=(A.sub.isovaleryl
I+A.sub.isovaleryl II+A.sub.isovaleryl
III).times.W.sub.S.times.P/(A.sub.S.times.W.sub.T).times.100%
Total content of acylation spiramycin (%)=(A.sub.acetyl
II+A.sub.Acetyl III+A.sub.propionyl II+A.sub.propionyl
III+A.sub.isobutyrylII+A.sub.isovaleryl I+A.sub.isobutyryl
III+A.sub.isovaleryl II+A.sub.isovaleryl
III).times.W.sub.S.times.P/(A.sub.S.times.W.sub.T).times.100%
Spiramycin III (%)=A.sub.spiral
II.times.W.sub.S.times.P/(A.sub.S.times.W.sub.T).times.100%
Unknown components
(%)=A.sub.WW.sub.S.times.P/(A.sub.S.times.W.sub.T).times.100%
Where:
[0116] W.sub.S--weight of standard sample, g; [0117] A.sub.S--peak
area of isovalerylspiramycin III in the standard sample; [0118]
W.sub.T--weight of testing sample, g; [0119] A.sub.W--total peak
area of unknown components in the testing sample; [0120] P--purity
of isovalerylspiramycin III in the testing sample.
Example 10
HPLC Detection of Levocarrimycin Component
[0121] Extract the eight batch fermentation broth of levocarrimycin
fermented by the levocarrimycin extraction process provided in
example 4 and the HPLC quantitative detection method provided in
example 9, the HPLC detection conditions of each component obtained
is shown as table 1 and the liquid chromatogram is shown as FIG.
5.
TABLE-US-00003 TABLE 1 the HPLC detection condition of eight
batches levocarrimycin components Percentage Average content % 1 2
3 4 5 6 7 8 value isv-III 35.71 34.40 34.80 32.19 35.71 35.80 35.59
35.44 34.96 isv-II 24.67 24.93 24.36 24.85 20.02 23.91 23.87 23.76
23.80 isv-I 2.30 2.94 4.07 3.18 3.46 2.90 3.40 3.00 3.16 bu-III
3.56 2.75 3.54 3.50 3.50 3.90 4.10 4.00 3.61 ibu-III -- -- -- -- --
-- -- -- -- bu-II 0.99 1.00 1.15 1.07 1.20 1.30 1.20 0.99 Ibu-II --
-- -- -- -- -- -- -- -- pr-III 7.91 8.09 7.65 8.19 8.24 8.40 8.70
8.50 8.21 pr-II 2.91 2.65 3.07 3.72 3.90 5.40 5.11 5.36 4.02 ac-III
1.50 1.19 1.07 0.96 1.14 1.36 1.68 1.29 1.27 ac-II 2.84 2.92 3.05
3.08 3.47 1.89 3.18 3.09 2.94 Total isv 62.68 62.27 63.23 60.22
59.19 62.61 62.86 62.20 61.91 Total acylation 82.39 80.87 81.61
80.82 80.51 84.76 86.93 85.64 82.94
[0122] The above detection is also conducted for the levocarrimycin
prepared in other examples in the present invention and the liquid
chromatogram obtained is as shown in FIG. 5.
Example 11
Determination of Specific Rotation of Levocarrimycin
[0123] Precisely weigh a proper amount of levocarrimycin prepared
in the examples of the present invention, add chloroform to
dissolve and dilute into solution of 0.02 g/ml chloroform, and
determine the specific rotation with D line (589.3 nm) of natrium
spectrum with a determination length of 1 dm, determination
temperature of 25.quadrature., polarimeter with reading to
0.0001.degree. after being calibrated.
TABLE-US-00004 TABLE 2 Investigation result of specific rotation
Example NO. 1 2 3 4 5 6 7 8 [.alpha.].sup.25 -52.degree.
-55.2.degree. -57.degree. -56.degree. -54.degree. -55.3.degree.
55.1.degree. 55.4.degree.
Example 12
Levocarrimycin Tablets (Calculated in 10000 Tablets)
TABLE-US-00005 [0124] Formulation: Levocarrimycin raw powder
provided 1000 g by example 4 Low-substituted hydroxy propyl 92.5 g
cellulose (5%) Sodium carboxymethyl starch (3%) 55.5 g Magnesium
stearate (1%) 18.5 g Starch Total weight-weight of other raw
adjuvant materials Total weight 1850 g
[0125] Preparation process: Weigh a proper amount of starch, dilute
it to a concentration of 15%, heat it to a paste, and make into
adhesive; make the main material carrimycin, adjuvant materials
starch, low-substituted hydroxy propyl cellulose, sodium
carboxymethyl starch and magnesium stearate pass through a 100-mesh
sieve respectively, and weigh the needed main and adjuvant
materials according to formulation; after mixing the carrimycin,
starch, low-substituted hydroxy propyl cellulose uniformly, add
starch paste of 15% starch concentration to make soft material,
granulation with 14-mesh sieve, dry at 50-60.quadrature., control
the moisture at 3.about.5%, straighten granules with 14-mesh sieve,
add sodium carboxymethyl starch, magnesium stearate and mix, then
determine the content of granules; calculate the weight of every
tablet according to content of granules, tablet (.PHI.9 mm retuse
punch), detect differences of weight of tablets; package the
qualified tablets after inspection.
Example 13
Levocarrimycin Capsules (Calculated in 10000 Capsules)
TABLE-US-00006 [0126] Formulation: Levocarrimycin raw powder in
example 4 1000 g Starch (for pharmaceutical purpose) 1080-weight of
carrimycin raw powder Pharmaceutical NO. 3 capsules 1000 capsules
Liquid paraffin 50 ml
[0127] Preparation process: Respectively weigh the main material
carrimycin and adjuvant material pharmaceutical starch according to
the process formulation, add in a mixer for full mixing for 1.5-2
hours; the obtained data of the sample content tested shall be in
basic accordance with theoretical data (weight in every capsule is
about 0.105 g), respectively put the qualified pharmaceutical No. 3
capsules and the well-mixed raw materials into a filling machine
according to operation requirements of the automatic capsule
filling machine, conduct difference inspection for filled capsules
(within .+-.10%, <0.3 g), and the dissolution shall meet the
requirements; put the qualified capsules into a polishing machine
and add liquid paraffin to conduct polishing for 15-20 min, and
then take the capsules out and inspect the finished product packing
boxes.
Example 14
Levocarrimycin Sugar-Coated Tablets (Calculated in 10000
Tablets)
[0128] Formula: Same as that of example 12
Preparation process: Operate as the method in example 12, put the
qualified core tablets into a sugar-coating pot, slowly put the
prepared syrup (concentration of 65-70%) into the pot, and then
raise the temperature to about 40.quadrature., add a proper amount
of talcum, and conduct forced air drying for 20-30 min; after the
tablets gain a sub-coat by repeating the above steps for several
times, conduct sugar coating for 15-20 min; after the tablets gain
a sugar coat put the prepared color paste into syrup and mix up,
and then pour it into the pot and mix for 15-20 min every time to
obtain the sugar-coated tablet.
Example 15
Levocarrimycin Dry Syrup (Calculated in 10000 Bags)
TABLE-US-00007 [0129] Formulation: Levocarrimycin raw powder in
1250 g example 4 Citric acid (0.5%) (citrate) 15 g Sucrose Total
weight-weight of other adjuvant materials Total weight 500 g
Pigment (curcumin) about 1 g
[0130] Preparation process: Respectively crush the carrimycin raw
powder, citric acid, sucrose with a high-speed airflow crusher into
granules in such a manner that 85% of which can pass through a
300-mesh sieve, and 15% pass through a 180-mesh sieve, respectively
weigh a proper amount of the crushed fine powder according to the
formulation and then mix them adequately for 1-1.5 hours; determine
its content, calculate the packing volume (theoretical packing
content is 500 mg every bag), and then put the mixture into a
forming-filling-sealing machine, and pack it with aluminized paper.
Pack the product according to the operation requirements of
packaging machine, with the difference of packing content limited
within +5%, inspect after packing, and then implement out packing
for the qualified.
Example 16
Levocarrimycin Enteric-Coated Tablets (Calculated in 10000
Tablets)
[0131] Formulation: Refer to example 12.
[0132] Preparation process: prepare the tablet cores according to
example 12; put the qualified tablet cores into a sugar coating
pot, use 60-70% syrup and talcum powder to coat three base coating
layers and then coat the isolation layer, add 10% zein alcohol
solution, dry for 10-15 min with rollover method, and then drop
diethyl phthalate, acetone, cellulose acetate phthalate and alcohol
solution, i.e. the enteric-coated solution into the pot and dry for
2-3 times and 10-15 min of one time with rollover method; after
conforming qualification in examination, conduct sugar coating
according to example 7.
Example 17
Levocarrimycin Gastric-Coated Tablets (Calculated in 10000
Tablets)
[0133] Formulation: Refer to example 12.
[0134] Preparation process: prepare the tablet cores according to
example 12; put the qualified tablet cores into a high-efficiency
coating machine and then prepare the qualified coating powder
(including fat soluble and water soluble) into coating solution
according to the requirements and then put the coating solution
into the colloid for crushing and filtering for use. Preheat the
high-efficiency coating pot filled with tablet cores, with rotation
speed controlled within 5-10 rpm and temperature of
45.about.60.quadrature., spray the coating solution into the pot
with aerosol sprayer (>300 meshes) and dry for 25-35 minutes,
conduct the process repeatedly for 8-12 times, until the coating is
uniform, and finally pack the qualified tablets after drying.
Example 18
Levocarrimycin Granules (Calculated in 10000 Bags)
TABLE-US-00008 [0135] Formulation: Levocarrimycin raw powder in
example 5 1250 g Sugared powder 20000 g Dextrin 9000 g 5% PVP-K30
Appropriate amount
[0136] Preparation process: screen the carrimycin raw powder,
powdered sugar and dextrin with a 120-mesh sieve, weigh carrimycin
raw powder, powdered sugar and dextrin according to the formulation
and mix them uniformly; made the above mixed uniform material into
soft material with 5% PVP-K30 mucilage; prepare the material into
granules with oscillating granulator, dry in 70.quadrature.,
straighten granules, and then pack them after being inspected
qualified.
Example 19
Levocarrimycin Freeze-Dried Powder Injection
[0137] Weigh 500 mg levocarrimycin raw powder prepared in example
6, mix it with adipic acid of equal mole, and then dissolve into 5
ml water to obtain light yellow transparent solution, with a pH
value of 4.6.about.5.6. Add 40 mg mannitol as freeze-dried
proppant, rapidly freeze for 9 h at a low temperature, and
freeze-dried to obtain light yellow loose lump. Dissolve it with 10
ml sterile water before use.
Example 20
Levocanimycin Freeze-Dried Powder Injection
[0138] Weigh 500 mg levocarrimycin raw powder prepared in example
4, mix it with citric acid of equal mole, and then dissolve into 5
ml water to obtain light yellow transparent solution, with a pH
value of 4.6.about.5.6. Add 40 mg mannitol as freeze-dried
proppant, rapidly freeze for 9 h at a low temperature, and
freeze-dried to obtain light yellow loose lump. Dissolve it with 10
ml sterile water before use.
Example 21
Levocarrimycin Freeze-Dried Powder Injection
[0139] Weigh 500 mg levocarrimycin raw powder prepared in example
5, mix it with maleic acid of equal mole, and then dissolve into 5
ml water to obtain light yellow transparent solution, with a pH
value of 4.6.about.5.6. Add 40 mg mannitol as freeze-dried
proppant, rapidly freeze for 9 h at a low temperature, and
freeze-dried to obtain light yellow loose lump. Dissolve it with 10
ml sterile water before use.
Example 22
Preparation of Levocarrimycin in which the Isovalerylspiramycin I
is the Crystal Compound of Levoisovalerylspiramycin I
[0140] Separate and purify levocarrimycin obtained in example
1.
Purification of levoisovalerylspiramycin I: Purify the sample
obtained in the preliminary separation with a preparative HPLC,
prepare chromatographic column with ODS, use acetonitrile and
ammonium acetate buffer solution as the mobile phase in a gradient
elute; record the separated UV spectrogram through UV detection,
and collect the target peaks of levoisovalerylspiramycin I
components: Chromatographic column: ODS preparative chromatographic
column; Mobile phase: Acetonitrile (A), 100 mM ammonium acetate
solution (B); Gradient condition: adopting linear gradient for
0.about.60 minutes, A is 25%.about.65%, and 61.about.90 minutes, A
65%-90%; Flow velocity: 260 ml/min; Injection volume: 10 mL;
Sampling concentration: 0.5 g/mL; Measurement wavelength: 231 nm;
Way of collecting: collection via UV triggering;
[0141] Collect the sample of levoisovalerylspiramycin I according
to the retention time (RT) 44.759 min of levoisovalerylspiramycin
I, then eliminate acetonitrile through rotary evaporation, extract
with 1 time amount of ethyl acetate, and eliminate the ethyl
acetate in the extract through rotary evaporation to obtain paste
sample; re-dissolve the paste sample with petroleum ether, and
eliminate the petroleum ether through rotary evaporation to obtain
the white solid powder of levoisovalerylspiramycin I.
[0142] Further recrystallize the white solid powder
levoisovalerylspiramycin I to obtain the crystal compound. The
method of recrystallization is as below:
(1) Dissolve the solid compound of levoisovalerylspiramycin I
obtained in example 1 in the mixed solvent of ethyl acetate,
absolute ethyl alcohol and anhydrous acetone, with the volume ratio
of ethyl acetate to absolute ethyl alcohol to anhydrous acetone in
the mixed solvent being 1:10:1; (2) Then add pure water and stir
the mixture simultaneously, and the volume of pure water added is
2.5 times of total volume of ethyl acetate, absolute ethyl alcohol
and anhydrous acetone; the adding velocity of water is 4 ml/min;
and the stirring rate when adding pure water is 30 rpm; (3) Cool to
5.quadrature. at a speed of 1.quadrature./h after adding pure
water, continue stirring at a speed of 10 rpm when cooling, to
obtain the crystal compound of levoisovalerylspiramycin I.
[0143] The X-ray powder diffraction of the crystal compound of
levoisovalerylspiramycin I measured by Cu--K.alpha. X-ray has
characteristic peaks of 2.theta. at 7.6.degree., 8.0.degree.,
10.0.degree., 11.4.degree., 16.4.degree., 17.0.degree.,
17.5.degree., 17.9.degree., 19.5.degree., 22.7.degree.,
23.7.degree. and 24.4.degree., and the spectrum of X-ray powder
diffraction is as shown in FIG. 6.
[0144] Eliminate the acetonitrile in the residual levocarrimycin
after separating and purifying levoisovalerylspiramycin I
components through rotary evaporation, then extract with 1 time
amount of ethyl acetate, and eliminate the ethyl acetate in the
extract through rotary evaporation to obtain paste sample;
re-dissolve the paste sample with petroleum ether, and eliminate
the petroleum ether through rotary evaporation to obtain the
levocarrimycin; then mix the levocarrimycin with the above crystal
compound of levoisovalerylspiramycin I to obtain the
levocarrimycin, in which the isovalerylspiramycin I is the crystal
compound of levoisovalerylspiramycin I.
Example 23
Preparation of Levocarrimycin in which the Isovalerylspiramycin I
is the Crystal Compound of Levoisovalerylspiramycin I
[0145] Besides the steps which are the same as those in example 22,
the difference in method of recrystallization is as below:
(1) Dissolve the solid compound of levoisovalerylspiramycin I in
the mixed solvent of ethyl acetate, absolute ethyl alcohol and
anhydrous acetone, with the volume ratio of ethyl acetate to
absolute ethyl alcohol to anhydrous acetone in the mixed solvent
being 1:10:1; (2) Then add pure water and stir the mixture
simultaneously, and the volume of pure water added is 9 times of
total volume of ethyl acetate, absolute ethyl alcohol and anhydrous
acetone; the adding velocity of water is 10 ml/min; and the
stirring rate when adding pure water is 60 rpm; (3) Cool to
15.quadrature. at a speed of 3.quadrature./h after addition of pure
water, and continue stirring at a speed of 10 rpm when cooling, to
obtain the crystal compound of levoisovalerylspiramycin I.
[0146] The X-ray powder diffraction of the crystal compound of
levoisovalerylspiramycin I measured by Cu--K.alpha. ray is similar
to that of FIG. 6.
Example 24
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
I is the Crystal compound of Levolevoisovalerylspiramycin I
[0147] Other operation steps are the same as example 22. What is
different is the recrystallization, as follows:
1. First, dissolve the solid compound of levoisovalerylspiramycin I
in the mixed solvent of ethyl acetate, absolute ethyl alcohol, and
anhydrous acetone with the volume ratio of ethyl acetate to
absolute ethyl alcohol to anhydrous acetone in the mixed solvent
being 1:5:0.8; 2. Then add pure water and stir the mixture
simultaneously, and the volume of pure water added is 7.5 times of
the total volume of ethyl acetate, absolute ethyl alcohol, and
anhydrous acetone; the adding velocity of water is 6 ml/min; and
the stirring rate when adding pure water is 40 rpm; 3. Cool to
10.quadrature. at a speed of 2.quadrature./h after addition of pure
water, and continue stirring at a speed of 15 rpm when cooling, to
obtain the crystal compound of levoisovalerylspiramycin I.
[0148] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin I is similar to that of FIG. 6.
Example 25
The Preparation of Levocarrimycin in which the Isovalcrylspiramycin
I is the Crystal Compound of Levoisovalerylspiramycin I
[0149] Other operation steps are the same as example 22. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
I in the mixed solvent of ethyl acetate, absolute ethyl alcohol,
and anhydrous acetone with the volume ratio of ethyl acetate to
absolute ethyl alcohol to anhydrous acetone in the mixed solvent
being 1:2:1; (2) Then add pure water and stir the mixture
simultaneously, and the volume of pure water added is 7.5 times of
the total volume of ethyl acetate, absolute ethyl alcohol, and
anhydrous acetone; the adding velocity of water is 8 ml/min; and
the stirring rate when adding pure water is 45 rpm; (3) Cool to
12.quadrature. at a speed of 2.5.quadrature./h after addition of
pure water, and continue stirring at a speed of 20 rpm when
cooling, to obtain the crystal compound of levoisovalerylspiramycin
I.
[0150] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin I is similar to FIG. 6.
Example 26
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
I is the Crystal Compound of Levoisovalerylspiramycin I
[0151] Other operation steps are the same as example 22. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
I in the mixed solvent of ethyl acetate, absolute ethyl alcohol,
and anhydrous acetone with the volume ratio of ethyl acetate to
absolute ethyl alcohol to anhydrous acetone in the mixed solvent
being 1:5:0.8; (2) Then add pure water and stir the mixture
simultaneously, and the volume of pure water added is 5 times of
the total volume of ethyl acetate, absolute ethyl alcohol, and
anhydrous acetone; the adding velocity of water is 7 ml/minute; and
the stirring rate when adding pure water is 60 revolutions/minutes;
(3) Cool to 12.quadrature. at a speed of 1.2.quadrature./h after
addition of pure water, and continue stirring at a speed of 15 rpm
when cooling, to obtain the crystal compound of
levoisovalerylspiramycin I.
[0152] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin I is similar to FIG. 6.
Example 27
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
II is the Crystal Compound of Levoisovalerylspiramycin II
[0153] Purify the levocarrimycin obtained in example 2. The
detailed operation steps are the same as example 22. What is
different is the sample of levoisovalerylspiramycin II is collected
according to the retention time RT 43.34 of
levoisovalerylspiramycin II.
[0154] Further recrystallize the white solid powder
levoisovalerylspiramycin II to obtain the crystal compound. The
method of recrystallization is as below:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
II obtained in example 2 in the mixed solvent of absolute methanol,
absolute ethyl alcohol, and anhydrous acetone with the volume ratio
of absolute methanol to absolute ethyl alcohol to anhydrous acetone
in the mixed solvent being 1:10:1; (2) Then add pure water and stir
the mixture simultaneously, and the volume of pure water added is
2.5 times of the total volume of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone; the adding velocity of water is 4
ml/min; and the stirring rate when adding pure water is 30 rpm; (3)
Cool to 5.quadrature. at a speed of 1.quadrature./h after addition
of pure water, and continue stirring at a speed of 10 rpm when
cooling, to obtain the crystal compound of levoisovalerylspiramycin
II.
[0155] The X-ray powder diffraction of the crystal compound of
levoisovalerylspiramycin II measured by Cu--K.alpha. X-ray has
characteristic peaks of 2.theta. at 10.0.degree., 11.6.degree.,
16.4.degree., 17.3.degree., 19.1.degree., 21.2.degree.,
22.1.degree., 22.7.degree., 26.4.degree., 26.9.degree.,
27.5.degree., and 31.5, and the spectrum of X-ray powder
diffraction is as shown in FIG. 7. Eliminate acetonitrile of
levocarrimycin which is purified and separated from the
compositions of levoisovalerylspiramycin III through rotary
evaporation, then extract the levocarrimycin with 1 times ethyl
acetate, and eliminate ethyl acetate in the extract through rotary
evaporation to obtain paste sample; redissolve the paste samples in
petroleum ether, and then eliminate petroleum ether through rotary
evaporation to obtain the levocarrimycin; then mix the
levocarrimycin and the above crystal compound of
levoisovalerylspiramycin II to make the levocarrimycin in which
isovalerylspiramycin II is the crystal compound of
levoisovalerylspiramycin II.
Example 28
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
II is the Crystal Compound of Levoisovalerylspiramycin II
[0156] Other operation steps are the same as example 27. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
II in the mixed solvent of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone with the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent being 1:10:0.8; (2) Then add pure water and stir the
mixture simultaneously. The volume of pure water added is 9 times
of the total volume of absolute methanol, absolute ethyl alcohol,
and anhydrous acetone; the adding velocity of water is 10 ml/min;
and the stirring rate when adding pure water is 60 rpm; (3) Cool to
15.quadrature. at a speed of 3.quadrature./h after addition of pure
water, and continue stirring at a speed of 10 rpm when cooling, to
obtain the crystal compound of levoisovalerylspiramycin II.
[0157] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin II is similar to FIG. 7.
Example 29
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
II is the Crystal Compound of Levoisovalerylspiramycin II
[0158] Other operation steps are the same as example 27. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
II in the mixed solvent of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone with the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent being 1:5:1; (2) Then add pure water and stir the
mixture simultaneously. The volume of pure water added is 7.5 times
of the total volume of absolute methanol, absolute ethyl alcohol,
and anhydrous acetone; the adding velocity of water is 6 ml/min;
and the stirring rate when adding pure water is 40 mp; (3) Cool to
10.quadrature. at a speed of 2.quadrature./h after addition of pure
water, and continue stirring at a speed of 15 rpm when cooling, to
obtain the crystal compound of levoisovalerylspiramycin II.
[0159] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin II is similar to FIG. 7.
Example 30
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
II is the Crystal Compound of Levoisovalerylspiramycin II
[0160] Other operation steps are the same as example 27. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
II in the mixed solvent of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone with the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent being 1:3:1; (2) Then add pure water and stir the
mixture simultaneously. The volume of pure water added is 7.5 times
of the total volume of absolute methanol, absolute ethyl alcohol,
and anhydrous acetone; the adding velocity of water is 8 ml/min;
and the stirring rate when adding pure water is 45 rpm; (3) Cool to
12.quadrature. at a speed of 2.5.quadrature./h after addition of
pure water, and continue stirring at a speed of 20 rpm when
cooling, to obtain the crystal compound of levoisovalerylspiramycin
II.
[0161] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin II is similar to FIG. 7.
Example 31
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
II is the Crystal Compound of Levoisovalerylspiramycin II
[0162] Other operation steps are the same as example 27. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
II in the mixed solvent of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone with the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent being is 1:6:0.8; (2) Then add pure water and stir
the mixture simultaneously. The volume of pure water added is 5
times of the total volume of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone; the adding velocity of water is 7
ml/min; and the stirring rate when adding pure water is 60 rpm; (3)
Cool to 12.quadrature. at a speed of 1.2.quadrature./h after
addition of pure water, and continue stirring at a speed of 15 rpm
when cooling, to obtain the crystal compound of
levoisovalerylspiramycin II.
[0163] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin II is similar to FIG. 7.
Example 32
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
III is the Crystal Compound of Levoisovalerylspiramycin III
[0164] Purify the levocarrimycin made in example 3. The detailed
operation steps are the same as example 22. Collect the samples of
levoisovalerylspiramycin III in accordance with the retention time
48.009 of levoisovalerylspiramycin III.
[0165] Further recrystallize the white solid powder
levoisovalerylspiramycin III to obtain the crystal compound. The
method of recrystallization is as below:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
III made in example 3 in the mixed solvent of absolute methanol,
absolute ethyl alcohol, and anhydrous acetone with the volume ratio
of absolute methanol to absolute ethyl alcohol to anhydrous acetone
in the mixed solvent being 1:10:1; (2) Then add pure water and stir
the mixture simultaneously, and the volume of pure water added is
2.5 times of the total volume of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone; the adding velocity of water is 4
ml/min; and the stirring rate when adding pure water is 30 rpm; (3)
Cool to 5.quadrature. at a speed of 1.quadrature./h after addition
of pure water, and continue stirring at a speed of 10 rpm when
cooling, to obtain the crystal compound of levoisovalerylspiramycin
III.
[0166] The X-ray powder diffraction of the crystal compound of
levoisovalerylspiramycin III measured by Cu--K.alpha. X-ray has
characteristic peaks of 2.theta. at 8.0.degree., 10.0.degree.,
11.2.degree., 11.7.degree., 16.4.degree., 19.1.degree.,
19.6.degree., 20.0.degree., 21.4.degree., 22.9.degree.,
23.6.degree., and 29.4.degree., and the spectrum of X-ray powder
diffraction is as shown in FIG. 8. Evaporate acetonitrile of
levocarrimycin purified and separated from the compositions of the
levoisovalerylspiramycin III through rotary evaporation, then
extract the levocarrimycin with 1 times ethyl acetate, and
evaporate ethyl acetate through rotary evaporation to obtain the
paste samples; redissolve the paste samples in petroleum ether, and
then evaporate the petroleum ether through rotary evaporation to
obtain the levocarrimycin; then mix the levocarrimycin and the
above crystal compound of levoisovalerylspiramycin III to obtain
the levocarrimycin in which the isovalerylspiramycin III is the
crystal compound of levoisovalerylspiramycin III.
Example 33
The Preparation of Levocarrimycin in which the Isovalerylspiramycin
III is the Crystal Compound of Levoisovalerylspiramycin III
[0167] Other operation steps are the same as example 32. What is
different is the recrystallization, as follows:
(1) First, dissolve the solid compound of levoisovalerylspiramycin
III in the mixed solvent of absolute methanol, absolute ethyl
alcohol, and anhydrous acetone with the volume ratio of absolute
methanol to absolute ethyl alcohol to anhydrous acetone in the
mixed solvent being 1:10:1; (2) Then add pure water with stirring.
The volume of pure water added is 9 times of the total volume of
absolute methanol, absolute ethyl alcohol, and anhydrous acetone;
the adding velocity of water is 10 ml/min; and the stirring rate
when adding pure water is 60 rpm; (3) Cool to 15.quadrature. at a
speed of 3.quadrature./h after addition of pure water, and continue
stirring at a speed of 10 rpm when cooling, to obtain the crystal
compound of levoisovalerylspiramycin III.
[0168] Measured through Cu--K.alpha. ray, the X-ray powder
diffraction spectrum of the crystal compound of
levoisovalerylspiramycin III is similar to FIG. 8.
Example 34
Preparation of Levocarrimycin in which the Isovalerylspiramycin III
is the Crystal Compound of Levoisovalerylspiramycin III
[0169] Other operating steps are the same as example 32. The
different ways of recrystallization are as follows:
(1) Dissolve the solid-state the compound of
levoisovalerylspiramycin III into the mixed solvent which composed
of absolute methanol, absolute ethyl alcohol and anhydrous acetone,
and volume ratio of absolute methanol to absolute ethyl alcohol to
anhydrous acetone in the mixed solvent is 1:5:0.8; (2) Add pure
water while stirring and the volume of added pure water is 7.5
times as much as the total volume of absolute methanol, absolute
ethyl alcohol and anhydrous acetone; pure water will be added at
the rate of 6 ml/min; the stirring rate for adding pure water is 40
rpm; (3) Cool it down to 10.quadrature. at the rate of
2.quadrature./h after adding pure water, and keep stirring while
cooling at the rate of 15 rpm; the crystal compound of
levoisovalerylspiramycin III is obtained.
[0170] The X-ray powder diffraction patterns of the crystal
compound of levoisovalerylspiramycin III measured by Cu--K.alpha.
ray are similar to FIG. 8.
Example 35
Preparation of Levocarrimycin in which the Isovalerylspiramycin III
is the Crystal Compound of Levoisovalerylspiramycin III
[0171] Other operating steps are the same as example 32, and the
different ways of recrystallization are as follows:
(1) Dissolve the solid-state compound of levoisovalerylspiramycin
III into the mixed solvent which composed of absolute methanol,
absolute ethyl alcohol and anhydrous acetone, and volume ratio of
absolute methanol to absolute ethyl alcohol to anhydrous acetone in
the mixed solvent is 1:2:1; (2) Add pure water while stirring and
the volume of added pure water is 7.5 times as much as the total
volume of absolute methanol, absolute ethyl alcohol and anhydrous
acetone; pure water will be added at the rate of 8 ml/min; the
stirring rate for adding pure water is 45 rpm; (3) Cool it down to
12.quadrature. at the rate of 2.5.quadrature./h after adding pure
water while stirring at the rate of 20 rpm, to obtain the crystal
compound of levoisovalerylspiramycin III.
[0172] The X-ray powder diffraction patterns of the crystal
compound of levoisovalerylspiramycin III measured by Cu--K.alpha.
ray are similar to FIG. 8.
Example 36
Preparation of Levocarrimycin in which the Isovalerylspiramycin III
is the Crystal Compound of Levoisovalerylspiramycin III
[0173] Other operating steps are the same as example 32, and the
different ways of recrystallization are as follows:
(1) Dissolve the solid-state compound of levoisovalerylspiramycin
III into the mixed solvent which composed of absolute methanol,
absolute ethyl alcohol and anhydrous acetone, and volume ratio of
absolute methanol to absolute ethyl alcohol to anhydrous acetone of
the mixed solvent is 1:5:0.8; (2) Add pure water while stirring and
the volume of added pure water is 5 times as much as the total
volume of absolute methanol, absolute ethyl alcohol and anhydrous
acetone; pure water will be added at the rate of 7 ml/min; the
stirring rate for adding pure water is 60 rpm; (3) Cool it down to
12.quadrature. at the rate of 1.2.quadrature./h after adding pure
water, while stirring at the rate of 15 rpm, to get the crystal
compound of levoisovalerylspiramycin III.
[0174] The X-ray powder diffraction patterns of the crystal
compound of levoisovalerylspiramycin III measured by Cu--K.alpha.
ray are similar to FIG. 8.
Example 37
Levocarrimycin Tablet Containing the Crystal Compound of
Levoisovalerylspiramycin I
[0175] The formulation and preparation method are the same as
example 12. The difference is that the levocarrimycin powder is the
levocarrimycin powder obtained in example 22, in which the
isovalerylspiramycin I is the crystal compound of
levoisovalerylspiramycin I.
Example 38
Levocarrimycin Tablet Containing the Crystal Compound of
Levoisovalerylspiramycin II
[0176] The formulation and preparation method are the same as
example 12. The difference is that the levocarrimycin powder is the
levocarrimycin powder obtained in example 27, in which the
isovalerylspiramycin II is the crystal compound of
levoisovalerylspiramycin II.
Example 39
Levocarrimycin Tablet Containing the Crystal Compound of
Levoisovalerylspiramycin III
[0177] The formulation and preparation method are the same as
example 12. The difference is that the levocarrimycin powder is the
levocarrimycin powder obtained in example 32, in which the
sovalerylspiramycin IIII is the crystal compound of
levoisovalerylspiramycin III.
Example 40
Levocarrimycin Capsule Containing the Crystal Compound of
Levoisovalerylspiramycin I
[0178] The formulation and preparation method are the same as
example 13, the difference is that the levocarrimycin powder is the
levocarrimycin powder obtained in example 23, in which the
isovalerylspiramycin I is the crystal compound of
levoisovalerylspiramycin I.
Example 41
Levocarrimycin Capsule Containing the Crystal Compound of
Levoisovalerylspiramycin II
[0179] The formulation and preparation method are the same as
example 13. The difference is that the levocarrimycin powder is the
levocarrimycin powder obtained in example 28, in which the
isovalerylspiramycin II is the crystal compound of
levoisovalerylspiramycin II.
Example 42
Levocarrimycin Capsule Containing the Crystal Compound of
Levoisovalerylspiramycin III
[0180] The formulation and preparation method are the same as
example 13. The difference is that the levocarrimycin powder is the
levocarrimycin powder obtained in example 33, in which the
isovalerylspiramycin III is the crystal compound of
levoisovalerylspiramycin III.
[0181] Adjuvant and preparation methods used for other preparations
of levocarrimycin, in which the isovalerylspiramycin I, II or III
is the crystal compound of levoisovalerylspiramycin I, II or III
are the same as above.
Test Example 1
In Vivo Pharmacodynamic
[0182] Test method: infectious bacteria liquid preparation: put the
bacteria liquid stored in the -80.quadrature. refrigerator into the
room for about 1 h at room temperature, and respectively draw 0.1
ml bacteria liquid of streptococcus pneumonia, streptococcus
pyogenes and enterococcus into 2 ml MH soup (add 10% inactivated
horse serum); inoculate the 2 ml MH soup with 0.1 ml bacteria
liquid of staphylococcus aureus according to the above method,
place the liquid into 37.quadrature. incubator for 18 h to get the
original bacteria liquid, dilute the original bacteria liquid with
5% gastric mucin, take 100% lethal bacteria amount if animal gets
infected as infectious bacteria liquid.
[0183] Oral administration is planned for clinical medication of
levocarrimycin, thus intragastric administration is selected for
levocarrimycin test. After intraperitoneal injection of 0.5 ml
lethal bacterial amount to the mice' abdominal cavity, the mice
appear following symptom, such as reduced activity, repose, hair
became loose, and so on. Clyster with 0.2 ml for every mouse after
infection 0.5-6 h, there is no adverse reaction. Observe animal
fatality within seven days and calculate the half protective dose
(EDO of drugs for infected mice and compare the protective effect
of drugs througe Bliss order.
[0184] In vivo test results are as shown in table 3 and 4
TABLE-US-00009 TABLE 3 Comparison of curative effect of four
antibiotics to the mice which abdominal cavity was infected by 6
strains streptococcus Challenging dose MIC (CFU/0.5 (.mu.g/
ED.sub.50 Test organism ml/mouse) Drugs ml) (mg/kg) Streptococcus
6.4 .times. 10.sup.4 Carrimycin 0.12 10.41 pneumonia.sub.3
Isovaleryl I is a crystal 0.12 8.99 compound Isovaleryl II is a
crystal 0.12 8.39 compound Isovaleryl III is a crystal 0.12 8.99
compound Azithromycin 0.5 18.29 Acetyl spiramycin 0.5 66.96
Erythrocin 1 85.08 Streptococcus 9.6 .times. 10.sup.4 Carrimycin
0.03 10.06 pneumonia.sub.18 Isovaleryl I is a crystal 0.03 9.94
compound Isovaleryl II is a crystal 0.03 9.08 compound Isovaleryl
III is a 0.03 8.98 crystal compound Azithromycin 0.06 14.87 Acetyl
spiramycin 0.06 37.93 Erythrocin 0.06 57.08 Streptococcus 8.8
.times. 10.sup.4 Carrimycin 0.12 16.02 pneumonia.sub.57 Isovaleryl
I is a crystal 0.06 13.60 compound Isovaleryl II is a crystal 0.06
13.86 compound Isovaleryl III is a 0.06 12.81 crystalline compound
Azithromycin 0.25 19.66 Acetyl spiramycin 1 398.01 Erythrocin 0.25
102.33 Streptococcus 6.9 .times. 10.sup.3 Carrimycin 0.12 26.30
pyogenes.sub.772 Isovaleryl I is a 0.06 26.15 crystalline compound
Isovaleryl II is a crystal 0.06 23.37 compound Isovaleryl III is a
crystal 0.06 23.37 compound Azithromycin 0.25 46.89 Acetyl
spiramycin 0.25 98.11 Erythrocin 0.5 101.33 Streptococcus 7.8
.times. 10.sup.4 Carrimycin 0.25 87.84 pyogenes.sub.102 Isovaleryl
I is a crystal 0.12 69.67 compound Isovaleryl II is a crystal 0.12
64.10 compound Isovaleryl III is a crystal 0.12 64.10 compound
Azithromycin 0.5 159.06 Acetyl spiramycin 0.5 227.07 Erythrocin 0.5
361.01 Streptococcus 4.9 .times. 10.sup.4 Carrimycin 0.25 68.48
pyogenes.sub.119 Isovaleryl I is a crystal 0.12 61.87 compound
Isovaleryl II is a crystal 0.12 59.91 compound Isovaleryl III is a
crystal 0.12 59.91 compound Azithromycin 0.5 98.98 Acetyl
spiramycin 0.5 117.53 Erythrocin 0.5 233.72
TABLE-US-00010 TABLE 4 Comparison of curative effect of 4
antibiotics to mice's abdominal cavity infected by enterococcus and
staphylococcus aureus Chal- lenging dose (CFU/0.5 MIC ED.sub.50
Test organism ml/mouse) Drugs (.mu.g/ml) (mg/kg)
Enterococcus.sub.32 5.4 .times. 10.sup.4 Carrimycin 0.5 89.29
Isovaleryl I is a crystal 0.5 85.15 compound Isovaleryl II is a
crystal 0.25 68.54 compound Isovaleryl III is a crystal 0.25 68.54
compound Azithromycin 1 146.51 Acetyl spiramycin 1 130.34
Erythrocin 2 175.23 Staphylococcus 5.2 .times. 10.sup.3 Carrimycin
0.5 31.98 aureus.sub.16 Isovaleryl I is a crystal 0.5 25.97
compound Isovaleryl II is a crystal 0.25 26.02 compound Isovaleryl
III is a crystal 0.25 26.02 compound Azithromycin 1 75.80 Acetyl
spiramycin 1 43.58 Erythrocin 1 82.36 Staphylococcus 5.8 .times.
10.sup.4 Carrimycin 0.5 31.50 aureus.sub.76 Isovaleryl I is a
crystal 0.5 26.50 compound Isovaleryl II is a crystal 0.25 25.16
compound Isovaleryl III is a crystal 0.25 25.16 compound
Azithromycin 1 58.79 Acetyl spiramycin 1 66.63 Erythrocin 1 64.17
Staphylococcus 4.8 .times. 10.sup.4 Carrimycin 2 120.35
aureus.sub.12 Isovaleryl I is a crystal 1 114.53 compound
Isovaleryl II is a crystal 1 109.59 compound Isovaleryl III is a
crystal 1 109.59 compound Azithromycin 4 217.36 Acetyl spiramycin
2048 >500 Erythrocin 256 266.11 Staphylococcus 4.2 .times.
10.sup.4 Carrimycin 1 59.30 aureus.sub.21 Isovaleryl I is a crystal
0.5 42.67 compound Isovaleryl II is a crystal 0.5 47.65 compound
Isovaleryl III is a crystal 0.5 47.65 compound Azithromycin 4
142.99 Acetyl spiramycin 2048 >500 Erythrocin 4 213.67
[0185] In vivo test results indicate that: the curative effect of
levocarrimycin to mice infected by 12 strains bacteria as shown in
table 3 and 4, the results shows that it has good protective
effect; and the levocarrimycin in which the isovalerylspiramycin I,
II or III is the crystal compound of levoisovalerylspiramycin I, II
or III shows better protective effect.
[0186] The same test is also conducted on the levocarrimycin or
levocarrimycin preparations prepared in other examples of the
present invention and the results are similar.
Test Example 2
In Vitro Pharmacodynamics
Determination of Clinical Isolates:
[0187] Test method: adopt two-fold plate dilution methods: pour the
quantitative melted agra culture-medium into the plate which
contains series drug concentration to mix with the liquid (add 5%
fiber-free goat blood into streptococcus and enterococcus to get
blood basal medium and add 7% fiber-free goat blood into bacillus
influenzae and gonococcus medium to get chocolate basal medium),
dilute the fresh-cultivated bacteria liquid into 106CFU/ml after
solidification, inoculate the cuvette agar of the levocarrimycin
obtained from example 4 and control group of azithromycin, acetyl
spiramycin and erythrocin through multipoint inoculating device,
culture them under 37.quadrature. for 18 h, put the gonococcus in a
incubator of 5% CO.sub.2 for 24 h; put legionella into a incubator
of 5% CO.sub.2 for 48 h; place anaerobe into a anaerobic box of
37.quadrature. for 48 h. Observe that the minimum concentration of
antibacterial drug that inhibits the growth of bacteria is the
minimal inhibitory concentration (MIC) and calculate drugs
MIC.sub.50 and MIC.sub.90 to be compared with the control
drugs.
[0188] Notes:
MIC.sub.50 inhibits 50% minimum Inhibitory concentration for the
growth of bacteria; MIC.sub.90 inhibits 90% minimum Inhibitory
concentration for the growth of bacteria. [0189] Test results are
as shown in the following table:
TABLE-US-00011 [0189] TABLE 5 Sensitive distribution of clinical
isolates by carrimycin Strain and number MIC scope MIC.sub.50
MIC.sub.90 of strain Drugs (.mu.g/ml) (.mu.g/ml) (.mu.g/ml)
Streptococcus Carrimycin 0.005->64 0.12 4 pneumonia Azithromycin
0.005->64 0.25 8 (112) Acetyl spiramycin 0.005->64 0.12
>64 Erythrocin 0.005->64 0.25 64 Streptococcus Carrimycin
0.06->64 0.25 64 pyogenes Azithromycin 0.25->64 0.5 >64
(93) Acetyl spiramycin 0.005->64 0.25 >64 Erythrocin
0.06->64 0.5 >64 Enterococcus Carrimycin 0.5->64 2 64
(106) Azithromycin 0.25->64 8 >64 Acetyl spiramycin
0.12->64 4 >64 Erythrocin 0.5->64 4 >64 Staphylococcus
Carrimycin 0.06->64 2 64 aureus Azithromycin 0.5->64 2 >64
(155) Acetyl spiramycin 0.12->64 64 >64 Erythrocin
0.12->64 1 >64 Staphylococcus Carrimycin 0.12->64 2 >64
epidermidis Azithromycin 0.12->64 8 >64 (115) Acetyl
spiramycin 0.03->64 64 >64 Erythrocin 0.06->64 8 >64
Bacillus influenzae Carrimycin 0.03-32 0.12 1 (37) Azithromycin
0.03->64 0.25 2 Acetyl spiramycin 0.03->64 0.12 4 Erythrocin
0.03->64 0.06 32 Gonococcus Carrimycin 0.12-16 2 8 (10)
Azithromycin 0.12-64 2 8 Acetyl spiramycin 0.12-64 4 8 Erythrocin
0.12-64 1 8
[0190] The same test is also conducted on levocarrimycin or
levocarrimycin preparations prepared in other examples of the
present invention and the results are similar.
Test Example 3
Determination of In Vitro Anti-chlamydia trachomatis and chlamydia
pneumoniae
Test Methods:
[0191] 1. Cultivate HEp-2 and McCoy cell line in a 96-pore cell
culture plate (Costar Company) respectively, 37.quadrature. and 5%
CO.sub.2, for 48 h to make monolayer cells. 2. Dilute the bacteria
to 10000.about.20000 ifu (inclusion bodies forming unit)/ml, adopt
0.1 ml/pore inoculation. Inoculate the McCoy cell culture plate
with chlamydia trachomatis serotype B/TW-5/OT and D/UW-3/Cx and
inoculate the HEp-2 cell culture plate with chlamydia pneumoniae
CWL-029. Firstly, suck the cell culture liquid in the 96-pore
culture plate, and then inocultate the plate by 0.1 ml/pore. Among
them, do not inoculate 4 pores of A11.about.D11 and 2 pores of C12
and D12 with bacteria. 3. After the inoculation, centrifuge the
96-pore cell culture plate with a centrifugal machine of
Beckman-Coulter Company, centrifugal force.times.1500 g,
centrifugal temperature 35.degree. C. and centrifugal time 60 min.
4. After the centrifugation, suck the inoculated chlamydia
trachomatis or chlamydia pneumoniae, and add 4 antibiotic drugs of
serial dilution into it respectively, namely the levocarrimycin
made in example 4 of the present invention; the acetylspiramycin,
erythrocin and azithromycin for control, 0.1 ml/pore. 5. Culture on
chlamydia trachomatis drug sensitive test plate at 37.quadrature.
and 5% CO.sub.2 for 48 h and on chlamydia pneumoniae drug sensitive
test plate for 72 h. After the culturing, suck antibiotic drug
solution and wash it twice with PBS (0.01M, pH 7.4), and then put
it in 100% methanol at room temperature for 15 min. 6. Indirect
immunofluorescent staining identification: add purified chlamydia
trachomatis resistant monoclonal antibody (N54 clone) and chlamydia
pneumoniae resistant monoclonal antibody (P33 clone), in chlamydia
trachomatis and chlamydia pneumoniae drug sensitive test plate
respectively, and incubate in SOW/pore and 37.quadrature. wet box
for 30 min. Wash the plate with a plate washer for 4 times, and
then add in rabbit anti-rat fluorescent antibody (Sigma Company),
SOW/pore. Incubate and wash the plate by using the same method and
at the same conditions. Add in mounting glycerol, 100 .mu.l/pore,
and observe the results under Nikon inverted fluorescence
microscope (Diaphot-200). 7. Definition of MIC: It refers to the
minimal antibiotic diluted concentration on that makes the growth
of Chlamdia Trachomatis or Chlamdia Pneumoniae incursion bodies in
96-pore test plates completely suppressed (No fluorescence staining
incursion is found in pores).
[0192] Test results are as follows:
TABLE-US-00012 TABLE 6 Minimal inhibitory concentration (MIC) in
vitro inhibition of four macrolide antibiotics against chlamydia
trachomatis and chlamydia pneumoniae Acetyl spiramycin Erythrocin
Azithromycin Carrimycin (AT-SPM) (EM) (AM) .mu.g/ml .mu.g/ml
.mu.g/ml .mu.g/ml Chlamydia 0.25 4 0.5 0.5 trachomatis B/TW-5/OT
Chlamydia 0.25 2 0.5 0.25 trachomatis D/UW-3/Cx Chlamydia 0.016 0.5
.ltoreq.0.016 0.032 pneumoniae CWL-029
[0193] 1. For chlamydia trachomatis serotype B/TW-5/OT, the MIC of
carrimycin is 0.250 .mu.g/ml, erythrocin and azithromycin (0.5
.mu.g/ml) comes second, and acetyl spiramycin (MIC is 4 .mu.g/ml)
comes last. [0194] 2. For chlamydia trachomatis serotype D/UW-3/Cx,
the in vitro effect of carrimycin and azithromycin is the same, MIC
is 0.25 .mu.g/ml, being sensitive; erythrocin (0.5 .mu.g/ml) comes
second and acetyl spiramycin (MIC is 2 .mu.g/ml) comes last. [0195]
3. For chlamydia pneumoniae CWL-029, the in vitro effect of
carrimycin and erythrocin is the most sensitive, MIC.ltoreq.0.016
.mu.g/ml, azithromycin (MIC is 0.032 .mu.g/ml) is more sensitive;
acetyl spiramycin (MIC is 0.5 .mu.g/ml) is poor. [0196] 4. In
general, the effect of the levocarrimycin of the present invention
against Chlamydia is better than other experimental drugs.
[0197] The same test is also conducted on the levocarrimycin or
levocarrimycin preparations prepared in other examples of the
present invention, and the results are similar.
Test Example 4
In Vitro Anti-Urea Plasma Urealyticum and Chlamydia pneumoniae
[0198] 1. Test method: add U-PPLO 0.8 ml in sterile 12-pore cell
culture plate (add 0.9 ml in bacteria liquid control pore and 1.0
ml in culture medium control pores).
[0199] 2. Add 10.sup.4CCU/ml Uu bacteria liquid 0.1 ml in every
experimental pores and the final amount of bacteria in the pores is
10.sup.3CCU/ml (do not add bacteria liquid in the control pore of
culture medium).
[0200] 3. Divide into 3 groups (100 .mu.g/ml, 10 .mu.g/ml and 1
.mu.g/ml antibiotic stocksolution), Add experimental antibiotics
(levocarrimycin in example 6 of the present invention, acetyl
spiramycin, erythrocin and azithromycin) in each experimental pores
with sterile Tip according to double degradation concentration
gradient: 100 .mu.l, 50 .mu.l, 25 .mu.l and 12.5 .mu.l. (Do not add
antibiotics in bacteria liquid control pore and culture medium
control pore. Meanwhile, the antibiotic control pore is
arranged.)
[0201] 4. Blend the above pores evenly, seal the culture plate with
adhesive tape and then culture in 37.quadrature. incubator.
[0202] 5. Abserve and record the growth of Uu on 17-24 h after the
test. When Uu bacteria liquid control pore shows positive growth,
the mimum antibiotic concentration that can inhibit the growth of
Uu is the MIC of the sample. The MIC after the test is the final
MIC (24 h), Determine the MIC of anti-urea plasma urealyticum
strains for 4 times and the results are as follows: [0203]
Carrimycin 0.025-0.125 .mu.g/ml, [0204] acetyl spiramycin 0.5
.mu.g/ml, [0205] erythrocin 5 .mu.g/ml, [0206] azithromycin
0.025-0.125 .mu.g/ml.
[0207] The above results show that carrimycin has good anti-Uu
function, which is similar to the function of azithromycin and
better than that of acetyl spiramycin. The anti-Uu function of
erythrocin in this test is the worst.
[0208] The same test is also conducted on levocarrimycin or
levocarrimycin preparations prepared in other examples of the
present invention and the results are similar.
* * * * *